BACKGROUND: A first step to combating antimicrobial resistance in enteric pathogens is to establish an objective assessment of antibiotic exposure. Our goal was to develop and evaluate a liquid chromatography-ion trap mass spectrometry (LC/MS) method to determine antibiotic exposure in patients with cholera. METHODS: A priority list for targeted LC/MS was generated from medication-vendor surveys in Bangladesh. A study of patients with and those without cholera was conducted to collect and analyze paired urine and stool samples. RESULTS: Among 845 patients, 11% (90) were Vibrio cholerae positive; among these 90 patients, analysis of stool specimens revealed ≥1 antibiotic in 86% and ≥2 antibiotics in 52%. Among 44 patients with cholera and paired urine and stool specimens, ≥1 antibiotic was detected in 98% and ≥2 antibiotics were detected in 84%, despite 55% self-reporting medication use. Compared with LC/MS, a low-cost antimicrobial detection bioassay lacked a sufficient negative predictive value (10%; 95% confidence interval, 6%-16%). Detection of guideline-recommended antibiotics in stool specimens did (for azithromycin; P = .040) and did not (for ciprofloxacin) correlate with V. cholerae suppression. A nonrecommended antibiotic (metronidazole) was associated with decreases in anaerobes (ie, Prevotella organisms; P < .001). CONCLUSION: These findings suggest that there may be no true negative control group when attempting to account for antibiotic exposure in settings like those in this study.