Purpose <p>In response to the increasing abuse of etomidate-related substances in China, this study developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of etomidate (ET), metomidate (MET), propoxate (PRO), isopropoxate (I-PRO), CF₃-etomidate (CF₃-ET), and their metabolite etomidate acid (ETA) in human nails. This method enabled the first investigation into their accumulation patterns in authentic samples, offering scientific support for forensic monitoring.</p> Methods <p>A 20&#xa0;mg portion of nail was cryogenically ground and extracted with methanol containing the internal standard. After vortex mixing, ultrasonication, and filtration, the extract was concentrated under nitrogen flow and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry.</p> Results <p>The limits of quantification (LOQs) for the target substances in nail samples ranged from 1 to 10 pg/mg. The calibration curves exhibited good linearity over the concentration range from the LOQ to 1000 pg/mg. All other evaluated validation parameters proved satisfactory. The validated method was applied to authentic nail samples to investigate the accumulation profiles of ET and its analogs. Analysis of the samples revealed that 52 tested positive. These positive cases showed a distinct accumulation pattern dominated by the parent drugs, with ETA as the sole metabolite detected.</p> Conclusion <p>This study developed and validated a reliable method for the simultaneous quantification of ET, its analogues, and its metabolite in human nails. Notably, it is the first to be applied to authentic samples to investigate their accumulation profiles, supporting the use of nail analysis in forensic toxicology and drug abuse monitoring.</p>