Background and Purpose: The co-use of opioids and other drugs is prevalent, both in a clinical setting and during opioid misuse. We investigated whether drug-drug interactions occur between oxycodone and xylazine, diazepam, etizolam, and methamphetamine using rat liver microsomes. Experimental Approach: Michaelis–Menten parameters (Km, Vmax), and intrinsic clearance (CLint) of oxymorphone and noroxycodone formation were determined. Inhibition Kis for xylazine, diazepam, and etizolam were assessed. Inhibition of oxycodone and dextromethorphan (probe substrate) metabolite formation by selective P450- and test-inhibitors was investigated. Key Results: Km values of oxymorphone (75 μM) and noroxycodone (74 μM) formation were similar. The Vmax of noroxycodone formation (1235 pmol/min/mg) was higher than oxymorphone (354 pmol/min/mg), leading to three times higher CLint of noroxycodone. Xylazine and etizolam were competitive inhibitors of oxymorphone (Ki = 0.5 and 9.5 μM, respectively) and noroxycodone (Ki = 2.6 and 14.6 μM, respectively) formation, while diazepam was a competitive inhibitor of oxymorphone formation (Ki = 3.7 μM) and a mixed inhibitor of noroxycodone formation (Ki = 8.3 μM). Xylazine, diazepam, and etizolam, as well selective P450-inhibitors, were confirmed to inhibit both CYP2D- and CYP3A-mediated pathways in rat liver microsomes. Methamphetamine was a moderate inhibitor of oxymorphone formation, and weak inhibitor of noroxycodone formation. Conclusion and Implications: Xylazine, diazepam, and etizolam are potent inhibitors of the formation of both primary oxycodone metabolites and leading to potential drug-drug interactions. Further, xylazine, diazepam, and etizolam could inhibit both CYP2D- and CYP3A-mediated pathways, leading to potentially altered drug metabolism of other opioids and substrates.