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Background Substance use is a leading cause of morbidity and mortality that is under-identified in medical practice. Objective The Tobacco, Alcohol, Prescription medication, and other Substance use (TAPS) Tool was developed to address the need for a brief screening and assessment instrument that includes all commonly used substances, and fits into clinical workflows. The goal of this study was to assess the performance of the TAPS Tool in primary care patients. Design Multi-site study conducted within the National Drug Abuse Treatment Clinical Trials Network, comparing the TAPS Tool against a reference standard measure. Setting Five adult primary care clinics. Participants 2,000 adult patients were consecutively recruited from clinic waiting areas. Measurements Interviewer- and self-administered versions of the TAPS Tool were compared to the reference standard modified Composite International Diagnostic Interview (CIDI), which measures problem use and substance use disorders (SUD). Results Interviewer- and self-administered versions of the TAPS Tool had similar diagnostic characteristics. For identifying problem use (at a cutoff of 1+), the TAPS Tool had sensitivity 0.93 (95% CI 0.90–0.95) and specificity 0.87 (95% CI 0.85–0.89) for tobacco, and sensitivity 0.74 (95% CI 0.70–0.78), specificity 0.79 (95% CI 0.76–0.81) for alcohol. For problem use of illicit and prescription drugs, sensitivity ranged from 0.82 (95% CI 0.76–0.87) for marijuana to 0.63 (95% CI 0.47–0.78) for sedatives, and specificity was 0.93 or higher. For identifying any SUD, sensitivity was lower, but a score of 2+ greatly increased the likelihood of having a SUD. Limitations Low prevalence of some drug classes led to poor precision in some estimates. Research assistants were not blinded to the participant’s TAPS Tool responses when they administered the CIDI. Conclusions In a diverse population of adult primary care patients, the TAPS Tool detected clinically relevant problem substance use. While it may also detect tobacco, alcohol, and marijuana use disorders, further refinement is needed before the TAPS Tool can be broadly recommended as a screener for SUD.