Injectable drug use presents a serious public health issue in Tunisia, increasing risks of infectious disease transmission and complicating monitoring efforts. Data on people who inject drugs (PWID) remain limited, hindering effective harm reduction strategies. In December 2023, 252 used syringes were collected from five sites in Tunis through a harm reduction program run by the Tunisian Association for Information and Orientation on AIDS and Addiction (ATIOST). Clean syringes had been distributed as part of a mobile syringe exchange service. Residual contents were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), following the generic protocol established by the European Syringe Collection and Analysis Project Enterprise (ESCAPE). Out of 252 syringes collected from five different sites, 82% contained at least one psychoactive substance; 45% contained multiple substances. Cocaine was most frequently detected (37.4%), followed by buprenorphine (27.64%) and heroin (12.4%). Eighteen percent of syringes had no detectable content. This method offers a quick way to gather data on drug use patterns within specific timeframes and geographic areas. The analysis can reveal variations in drug use trends through comparative case studies in countries within the European Neighbourhood Policy (ENP-South) region, thereby assisting in the adaptation of prevention and harm reduction strategies.