The ongoing epidemic of prescription opioid abuse in the United States has prompted interest in semi-synthetic opioids in the federal workplace drug testing program. This study characterized the metabolism and disposition of oxycodone (OC) in human urine. Twelve healthy adults were administered a single oral 20 mg dose of OC in a controlled clinical setting. Urine specimens were collected at timed intervals up to 52 h and analyzed by liquid chromatography-tandem mass spectrometry (limit of quantitation: 50 ng/mL) for OC, oxymorphone (OM), noroxycodone (NOC) and noroxymorphone (NOM) with and without enzymatic hydrolysis. OC and NOC appeared in urine within 2 h, followed by OM and NOM. Peak concentrations of OC and metabolites occurred between 3 and 19 h. Mean peak concentrations in hydrolyzed urine were in the following order: NOC > OC > OM > NOM. Only OM appeared to be excreted extensively as a conjugated metabolite. OC concentrations declined more quickly than NOC and OM. At a cutoff concentration of 50 ng/mL, detection times were approximately 30 h for OC and 40 h for NOC and OM. Some specimens did not contain OC, but most contained NOC, thereby facilitating interpretation that OC was the administered drug; however, five specimens contained only OM. These data provide information that should facilitate the selection of appropriate test parameters for OC in urine and assist in the interpretation of test results.