In January 2009, rodenticide bait (Ramik Green pellets) containing the active ingredient diphacinone was aerially applied to Lehua Island. Reported herein are the results of laboratory analyses to determine diphacinone concentrations in samples of seawater, fillet of fish, soft tissue of limpets (opihi), whole-body crabs, and soil collected from Lehua Island, Kauai County, Hawaii, after aerial application of the rodenticide bait. Diphacinone was specifically chosen because of its low toxicity to nontarget organisms. Its use on Lehua Island is the second time it has ever been used for an aerial application to eradicate rodents. Testing of the Lehua Island samples for diphacinone utilized high-performance liquid chromatography with photodiode array detection. No detectable concentrations of diphacinone were found in any of the samples from Lehua Island. The limits of detection for diphacinone were 0.4 nanograms per milliliter (parts per billion) seawater, 15 nanograms per gram (dry weight) soil, 20 nanograms per gram (parts per billion) fish fillet, 13 nanograms per gram whole crab, and 34 nanograms per gram soft tissue limpet.