A selective, sensitive, rugged and high throughput high performance liquid chromatography tandem mass spectrometric method was developed for the estimation of Emtricitabine in human urine using Abacavir as an internal standard (IS). Emtricitabine and abacavir were extracted from urine by solid phase extraction using Water Oasis HLB cartridges. The samples were chromatographed on Hypurity Advance, 50 x 2.1, 5μ column using a mobile phase consisting 5mM ammonium acetate: Acetonitrile: Methanol: (30:30:40 v/v). The chromatographic separation is achieved in 2.6 min. The method was validated over a concentration range of 0.50 μg/mL to 80.00 μg/mL. Method was validated for its sensitivity, selectivity, accuracy and precision, matrix effect, recovery and various stabilities. The validated method was used for analysis of urine samples. As per literature approximately 86% of emtricitabine is recovered in the urine unchanged hence urine analysis can serve as a useful tool to monitor patient adherence in HIV treatment.