Streptomycin is an aminoglycoside antibiotic used in apiculture to protect bees against a variety of brood diseases. Brazilian authorities have included it in the National Regulatory Monitoring Program for honey production. A simple and reliable method using liquid chromatography-tandem mass spectrometry has been developed and validated for the determination of streptomycin in honey. The chromatography separation was performed on a Gemini 5 microm C18 (50 mm x 2 mm) column using 5mM heptafluorbutiric acid/acetonitrile (85:15) as the mobile phase at a flow rate of 0.2 mLmin(-1). The detection of the analyte was achieved by positive ionization electrospray in multiple reaction-monitoring modes. Two characteristic transitions were monitored for streptomycin. Some analytical parameters were validated according to the guidelines laid down by European Commission Decision 2002/657/EC: decision limit, detection capability, recovery, precision and ruggedness. The recoveries of streptomycin from honey fortified at 2.5, 10, 15 and 20 microgkg(-1) levels are around 100%. The decision limit and detection capability of streptomycin was 3 microgkg(-1) and 4.7 microgkg(-1) respectively.