Tolperisone and etodolac were proven to have synergistic effect for patients of acute low back pain associated with musculoskeletal spasm. In this work, a specific, highly sensitive and reproducible analytical method was developed and validated for the simultaneous determination of tolperisone and etodolac in human plasma using liquid chromatography-tandem mass spectrometric technique. Liquid-liquid extraction was optimized for sample preparation. Zorbax C 8 column (3.5 μm, 50 × 4.6 mm) was used, carrying a mobile phase mixture of 10.0 mM ammonium formate:acetonitrile (40:60, v/v) pH 3.8, running in an isocratic mode. Chlorzoxazone acted as an internal standard. Sample volume of injection was 5.0 μL, and analysis was achieved within 2.5 min. Detection and quantitation were performed by electrospray ionization mass spectrometry using the multiple-reaction monitoring mode. The proposed method could determine the analytes in the range of concentration 0.5-200.0 ng mL −1 for tolperisone and 0.05-20.0 μg mL −1 for etodolac. Findings of inter-and intraday precisions were ≤12.3% with accuracy of ±5.0%. Pharmacokinetics study for the two drugs after oral administration of healthy human volunteers was achieved with the aid of application of the developed study.