In spite of the benefits of buprenorphine-naloxone co-formulation (BNX) in opioid maintenance treatment, the naloxone component has not prevented parenteral use of BNX. Current laboratory methods are not sufficient to differentiate between therapeutic and illicit use of buprenorphine, and little is known about urine naloxone concentrations. Measurement of urine naloxone, together with buprenorphine and norbuprenorphine, might help to determine the naloxone source and administration route. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for this purpose. Naloxone, buprenorphine, and norbuprenorphine total concentrations were measured in urine samples from opioid-dependent patients before and during stable and unstable phases of maintenance treatment with BNX. The limit of quantification in urine was 1.0 µg/L for naloxone, buprenorphine and norbuprenorphine. Before treatment, all samples contained buprenorphine but the median naloxone concentration was 0 µg/L. During the maintenance treatment with BNX all urine samples were positive for naloxone, buprenorphine and norbuprenorphine. The naloxone concentration at a stable phase of treatment (median 60 µg/L, range 5–200 µg/L) was not different from the naloxone concentration at an unstable phase (70 µg/L, 10–1700 µg/L). Applying an upper limit of 200 µg/L to the sample, the median naloxone/buprenorphine ratio was higher in the high than in the low naloxone concentration group (0.9 vs 0.3, respectively). This study suggests that naloxone in urine can act as an indicator of compliance with BNX. Parenteral use of BNX was associated with a high naloxone/buprenorphine ratio. Negative naloxone with positive buprenorphine suggests the use/abuse of buprenorphine alone. Copyright © 2013 John Wiley & Sons, Ltd.