This study of 20,089 urine specimens from chronic pain patients provided a unique opportunity to evaluate the prevalence of prescription opiates and metabolites, assess the usefulness of inclusion of normetabolites in the test panel, and compare opiate and oxycodone screening results to liquid chromatography with tandem mass spectrometry (LC-MS-MS) results. All specimens were screened by an opiate [enzyme-linked immunosorbent assay (ELISA), 100 ng/mL] and oxycodone assay [ELISA, 100 ng/mL or enzyme immunoassay (EIA), 50 ng/mL] and simultaneously tested by LC-MS-MS [limit of quantitation (LOQ) = 50 ng/mL] for 10 opiate analytes (codeine, norcodeine, morphine, hydrocodone, dihydrocodeine, norhydrocodone, hydromorphone, oxycodone, noroxycodone, and oxymorphone). Approximately two-thirds of the specimens were positive for one or more opiate analytes. The number of analytes detected in each specimen varied from 1 to 8 with 3 (34.8%) being most prevalent. Hydrocodone and oxycodone (in combination with metabolites) were most prevalent followed by morphine. Norcodeine was only infrequently detected whereas the prevalence of norhydrocodone and noroxycodone was approximately equal to the prevalence of the parent drug. A substantial number of specimens were identified that contained norhydrocodone (n = 943) or noroxycodone (n = 702) but not the parent drug, thereby establishing their interpretative value as biomarkers of parent drug use. Comparison of the two oxycodone screening assays revealed that the oxycodone ELISA had broader cross-reactivity with opiate analytes, and the oxycodone EIA was more specific for oxycodone. Specimens containing only norhydrocodone were best detected with the opiate ELISA whereas noroxycodone (only) specimens were best detected by the oxycodone EIA.