A simple and rapid reversed-phase high performance liquid chromatography (HPLC) assay for determination of tolperisone concentration in human plasma was developed and validated. Using prednisolone as an internal standard (IS), 1.0 ml plasma samples were extracted with a mixture of methyl tert. butyl ether and dichloromethane (70:30, v:v). Analysis was performed using Atlantis dC18 column with a mobile phase composed of 0.05 M (mono basic) potassium phosphate (pH=3.0) and acetonitrile (70:30, v:v). The eluent was monitored spectrophotometrically at 262 nm. No interference with tolperisone or IS peaks by extracted blank plasma components or commonly used drugs was observed. The relationship of tolperisone concentration and peak area ratio of tolperisone / IS was linear (R2 ≥ 0.9961) in the range of 10 – 800 ng/ml, the intra and inter-day coefficient of variations were ≤ 4.1% and ≤5.7%, respectively with a corresponding bias of ±11.4% and ±8.4%, respectively. Mean extraction recovery of tolperisone and the IS were 95% and 83%, respectively. The method was applied to assess the stability of tolperisone under various conditions encountered in the clinical laboratory. Tolperisone stability in processed samples stored at room temperature for 24 hours or at -20 °C for 48 hours, in unprocessed samples stored for 24 hours at room temperature or for 8 weeks at -20 °C, and after 3 freeze and thaw cycles was ≥ 91%.