Drug–drug interactions (DDIs) are a serious problem in the healthcare system, leading to excess healthcare utilization and costs. We conducted a second prospective randomized, controlled trial to further establish the real-world clinical utility of a novel assay that objectively identifies potentially serious DDIs in real-world patients. Re-recruiting primary care physicians (PCPs) from our first randomized, controlled, simulated-patients study on DDIs, we experimentally introduced a definitive, urine-based mass spectrometry test intervention that the physicians could use when caring for their eligible patients. Patients were eligible if taking four or more prescription medications or suspected of taking other non-prescribed substances with potential medication interactions. The primary outcome was whether DDI testing changed clinical care. We explored a secondary outcome to see if the change in practice improved symptoms in patients with potential DDIs. A total of 169 control and 162 intervention patients were enrolled in the study, and their medical records were abstracted. In real-world patients, intervention physicians identified and/or treated a DDI at 3.0x the rate in their patient population compared to controls (21.6% vs. 7.1%, p < 0.001). Intervention physicians were more likely to discontinue or adjust the interacting agent compared to controls (62.9% vs. 8.3%, p = 0.001), and patient-reported symptoms also significantly declined (29.6% vs. 20.1%, p = 0.045). These results were nearly identical to concurrent measurements that used simulated patients, wherein intervention was more likely to both make a DDI diagnosis (56.3% vs. 21.6%, p < 0.001) and stop the interacting medications (58.3% versus 26.6%, p < 0.001). Bringing a new diagnostic test to market, particularly for an under-recognized clinical problem, requires robust data on both clinical validity and clinical utility. The results of this follow-up study showed that the use of DDI testing in real-world patients significantly improved (1) primary care patient management of drug interactions and (2) patient outcomes.