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Mescaline, the primary psychoactive chemical in peyote cactus, has been consumed for thousands of years in ancient religious ceremonies. The US military wanted to determine if mescaline intake was a problem for personnel readiness. Twenty thousand seventeen urine specimens negative for cannabinoids, cocaine, opiates, and amphetamines were tested for mescaline with the Randox Drugs of Abuse V (DOA-V) biochip array immunoassay at the manufacturer's recommended cutoff of 6 mcg/L. A sensitive and specific method for mescaline quantification in urine was developed and fully validated. Extracted analytes were derivatized with pentafluoropropionic anhydride and pentafluoropropanol and quantified by gas chromatography–mass spectrometry (GC/MS) with electron impact ionization. Standard curves, using linear least squares regression with 1/x2 weighting, were linear from 1 to 250 mcg/L with coefficients of determination >0.994. Intra- and inter-assay imprecision was <4.4 coefficient of variation (%CV), with accuracies >90.4%. Mean extraction efficiencies were >92.0% across the linear range. This fully validated method was applied for the confirmation of urinary mescaline in 526 presumptive-positive specimens and 198 randomly selected presumptive-negative specimens at the manufacturer's 6 mcg/L cutoff. No specimen confirmed positive at the GC/MS limit of quantification of 1 mcg/L. Results indicated that during this time frame, there was insufficient mescaline drug use in the military to warrant routine screening in the drug testing program. However, mescaline stability, although assessed, could have contributed to lower prevalence. We also present a validated GC/MS method for mescaline quantification in urine for reliable confirmation of suspected mescaline intake.