Opioid use disorder poses a significant global health challenge, necessitating comprehensive management strategies. Integral to this approach are laboratory assessments, pivotal for confirming diagnosis, monitoring adherence, and evaluating complications. The testing process encompasses screening methods, primarily immunoassays and thin layer chromatography, and confirmatory techniques, including gas chromatography, liquid chromatography coupled with mass spectrometry, and innovative approaches like LC-MS/MS, ultra-high-performance liquid chromatography, and capillary electrophoresis. Notably, urine serves as the most common biological matrix for testing. Detection of opioids in various specimens relies on individual opioid metabolism. Beyond drug detection, laboratories contribute to assessing biochemical parameters, including liver and kidney functions, and identifying nutritional deficiencies. Given the heightened risk of viral infections, such as HIV and hepatitis C, within the opioid-using population, laboratories play a critical role in diagnosing and treating these consequential complications.