Urine drug testing (UDT) is a critical tool used in medical, forensic, and occupational settings, but interpreting results can be challenging. We performed a study to assess the ability of health care professionals to interpret UDT results accurately. In total, 911 clinical and laboratory professionals in the United States and Canada responded to a survey with questions gauging expertise in UDT interpretation. Responses were analyzed to identify knowledge gaps. Toxicologists and laboratory PhD scientists performed well, with means of 4.82 and 4.63 questions answered correctly (out of 6 possible), respectively. Physicians specializing in pathology, emergency medicine, primary care, and internal medicine, however, displayed concerning knowledge gaps, as did laboratorians with nondoctoral degrees. Experience and training correlated with interpretation accuracy. Identification of simulated compliance as well as understanding opioid exposure, metabolism, and immunoassay cross-reactivity were among the most clinicallysignificant knowledge gaps. More than 30% of survey respondents indicated that they would seek UDT information from the internet or peers rather than clinical or laboratory experts. The study highlighted the need for targeted education and better collaboration between clinical and laboratory experts and other health care professionals to ensure that when physicians order UDT, they can accurately interpret results and reduce harm.