Background: Despite the ample interest in the measurement of substance abuse and dependence, obtaining biological samples from participants as a means to validate a scale is considered time and cost intensive and is, subsequently, largely overlooked. Objectives: To report the psychometric properties of the drug use disorder (DUD) questionnaire including oral fluid and blood sample screening indicators measuring the three most commonly used illicit substances--marijuana, cocaine, and extramedicinal painkillers. Subjects: Participants were a subset ("N" = 2,702) of the 2007 U.S. National Roadside Survey that was administered to daytime and nighttime weekend drivers in the 48 contiguous states to examine the prevalence of substance use and misuse. Measures: Participants completed demographic and substance use questions as well as the DUD--a 12-item measure assessing substance abuse and dependence. Participants could potentially have completed the DUD three times for each of the three substances. Subscales of abuse and dependence were created using Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition Text Revision [DSM-IV-TR]) criteria of these diagnoses. Results: The DUD displayed adequate internal consistency on both subscales of substance abuse and dependence (Cronbach's alpha ranging from 0.71 to 0.84 and 0.83 to 0.92, respectively). The DUD also demonstrated construct validity in comparison to biological markers of each substance. Conclusions: The DUD is a biologically validated instrument that is both easy to utilize and may have valuable implications as a research tool among both clinical and nonclinical populations. (Contains 5 tables.)