Pheromones guide adult sea lamprey (Petromyzon marinus) to suitable spawning streams and mates, and therefore, when quantified, can be used to assess population size and guide management. Here, we present an efficient sample preparation method where 100 mL of river water was spiked with deuterated pheromone as an internal standard and underwent rapid field-based SPE and elution in the field. The combination of field extraction with laboratory UPLC-MS/MS reduced the sample consumption from 1 to 0.1 L, decreased the sample process time from more than 1 h to 10 min, and increased the precision and accuracy. The sensitivity was improved more than one order of magnitude compared with the previous method. The influences of experimental conditions were assessed to optimize the separation and peak shapes. The analytical method has been validated by studies of stability, selectivity, precision, and linearity and by the determination of the limits of detection and quantification. The method was used to quantify pheromone concentration from five streams tributary to Lake Ontario and to estimate that the environmental half-life of 3kPZS is about 26 h.