The role of unregulated and inappropriate dispensing, and use of antibiotics remains significant in the development of antimicrobial resistance in infectious disease endemic regions of developing countries. The exposure to antibiotics from unfamiliar and unsuspecting sources such as drinking water and food, and adulterated herbal medicines remains a cause for concern. A sensitive SPE-LC-MS/MS method was developed and validated for the quantification and qualification of 12 antibiotics, including amoxicillin, clavulanic acid, metronidazole, ampicillin, cefuroxime, tetracycline, ceftriaxone, sulphamethoxazole, trimethoprim, ciprofloxacin, benzylpenicillin, and erythromycin, in the urine of healthy volunteers. The method was linear (r(2) > 0.98) within the concentration range 50-5000 ngmL(-1) for all the analytes. Instrument precision of 8-27% and 4-21% at 100 and 1000 ngmL(-1) levels were demonstrated. High mean recoveries between 71 and 125% with minimal variations were obtained for all compounds in the accuracy study. Limits of detection and quantification ranged between 70.3-271.0 ngmL(-1) and 213-821 ngmL(-1) respectively. The validated method successfully detected and quantified 9 of the 12 analytes, with the exception of clavulanic acid, cefuroxime, and benzylpenicillin. Most of the samples contained one analyte (52, 86.7%), with a handful containing two (7, 11.7%) and three analytes (1, 1.7%). Ciprofloxacin was the modal analyte detected (17, 24.6%), with amoxicillin and trimethoprim recording the average lowest (22.76 x 10(3) ngmL(-1)) and highest concentrations (255.47 x 10(3) ngmL(-1)) respectively. The developed method is a useful tool for non-invasive monitoring of consumption and the irrational use of antibiotics in microbial resistant-prone regions of the world.