Piscicide applications in riverine environments are complicated by the advection and dispersion of the piscicide by the flowing water. Proper deactivation of the fish toxin is required outside of the treatment reach to ensure that there is minimal collateral damage to fisheries downstream or in connecting and adjacent water bodies. In urban settings and highly managed waterways, further complications arise from the influence of industrial intakes and outfalls, stormwater outfalls, lock and dam operations, and general unsteady flow conditions. These complications affect the local hydrodynamics and ultimately the transport and fate of the piscicide. This report presents two techniques using Rhodamine WT dye for real-time tracking of a piscicide plume—or any passive contaminant—in rivers and waterways in natural and urban settings. Passive contaminants are those that are present in such low concentration that there is no effect (such as buoyancy) on the fluid dynamics of the receiving water body. These methods, when combined with data logging and archiving, allow for visualization and documentation of the application and deactivation process. Real-time tracking and documentation of rotenone applications in rivers and urban waterways was accomplished by encasing the rotenone plume in a plume of Rhodamine WT dye and using vessel-mounted submersible fluorometers together with acoustic Doppler current profilers (ADCP) and global positioning system (GPS) receivers to track the dye and map the water currents responsible for advection and dispersion. In this study, two methods were used to track rotenone plumes: (1) simultaneous injection of dye with rotenone and (2) delineation of the upstream and downstream boundaries of the treatment zone with dye. All data were logged and displayed on a shipboard laptop computer, so that survey personnel provided real-time feedback about the extent of the rotenone plume to rotenone application and deactivation personnel. Further, these strategies facilitate adjustment of rotenone application and deactivation strategies in real time if necessary based on the observed advection and dispersion of the rotenone plume. Two large-scale and complex applications of rotenone in the Chicago Area Waterway System (CAWS) in 2009 and 2010 to combat invasive Asian carp are documented in this report. The application in Chicago Sanitary and Ship Canal (CSSC) in December 2009 involved more than 1,800 gallons of rotenone injected at multiple stations through a 6.2-mile reach of the canal near Lockport, Illinois. The rotenone plume was encased in Rhodamine WT dye so that two survey boats provided real-time feedback to shore personnel regarding the plume extent as it advected downstream. Real-time tracking of the rotenone was essential in this large-scale application because of the multistage injection strategy and the numerous deactivation points required to minimize collateral damage to fisheries in surrounding and receiving water bodies. All timing of application and deactivation operations relied on dye tracking. A second application of rotenone in May 2010 to the Little Calumet River near O’Brien Lock and Dam (Illinois) provided another opportunity for dye-tracking support operations; however, application and deactivation strategies were designed considering zero-flow conditions within the reach of interest. Therefore, dye was injected at the upstream and downstream boundaries of the rotenone application reach and was used to track movement of water in and out of a treatment reach, allowing proper deactivation to occur and avoiding unnecessary damage to fisheries downstream. The data collected during the real-time tracking operations for both applications allowed full documentation of the rotenone treatment for archival purposes and provided information for future applications. The methods presented in this report for real-time tracking and documentation of piscicide applications in riverine environments worked exceptionally well and allowed the multiagency Asian Carp Rapid Response Workgroup to carry out large-scale rotenone applications in urban waterways in an environmentally responsible manner with minimal collateral damage to fisheries outside the treatment reach. Traveltime information extracted from the boat-mounted and fixed-position fluorometers agrees well with empirical predictions from a preliminary dye study (mock rotenone injection) on this system completed in November 2009 on the CSSC and with previously published methods for estimating traveltimes of the peak, leading edge, and trailing edge of the plume. Although the rotenone application strategy called for zero-flow conditions on the Little Calumet River in 2010, downstream advection of treated water did occur, and dye tracing combined with velocity mapping allowed this advection to be documented and exposed the unique hydrodynamics and mixing within this reach. The large volumes of data collected during the operations allow documentation and visualization of the rotenone applications, thus providing feedback to planners and archival of the treatments for future reference. The methods developed in this report are directly transferrable to piscicide applications in water bodies in other locations, including rivers, ponds, or lakes, and can be used for real-time tracking of any passive contaminant that may enter a water body.