Laboratory methods for differentiating intravenous substance abuse from medical oral or sublingual use are lacking. The disaccharides lactose and sucrose are common excipients in both medical preparations and illicit drugs. If substances are abused intravenously, the disaccharides are excreted unchanged in urine. This study evaluated whether urine lactose indicates recent intravenous abuse of substances with special reference to buprenorphine (BPN). Samples were attained from 21 patients in assessment for opioid maintenance treatment (OMT) and from 29 patients already enrolled in OMT. Patients were divided in two groups. Group 1 patients reported intravenous abuse of BPN within 48 hours and Group 2 more than 48 hours since the last injection. The urine samples were assayed for lactose using an enzymatic method. To verify intravenous abuse, examination of needle marks and mass spectrometric analysis of abused substances was performed. Urine lactose concentration was significantly higher in Group 1 (86 mg/L) than in Group 2 (24 mg/L). All samples with lactose concentration more than 140 mg/L were given by the Group 1 patients, who all had recent injection marks. A significant inverse correlation was found between the time from last injection and the urine lactose concentration within the 48‐hour post‐injection period. Elevated lactose concentrations in urine could indicate intravenous abuse of substances containing lactose. The co‐occurrence of higher than 140 mg/L of lactose in urine by means of enzymatic assay verified by fresh puncture sites is a potential indicator of recent intravenous abuse of BPN.