A simple reverse phase high-performance liquid chromatography (HPLC) method was developed and validated for the quantitative determination of sparfloxacin (SPFX) in the bulk material, pharmaceutical formulation and human serum. Purospher Start C18 (25 cm × 4.6 mm, 5 µm) and Discovery C18 (25 cm × 4.6 mm, 5 µm) columns were used. The mobile phase, methanol, water and acetonitril (60:30:10 v/v/v pH 2.70 adjusted by phosphoric acid), was delivered at a flow rate of 1.0 mL min-1, eluent was monitored by ultra-violet (UV) detector at 290 nm. Gemifloxacin (GFX) was used as an internal standard. The proposed method is specific, accurate (98.11 to 102.83%), precise (intra and inter-day variations were 0.108 to 0.712% and 0.013 to 0.575%) and linearity was within the desired ranges of 2.5 to 100 µg mL-1 concentration having r2 > 0.998. All the results were correlated through analysis of variance (ANOVA) and Student’s t-test. The limit of detection (LOD) and limit of quantification (LOQ) were 0.0009 to 0.0064 and 0.0028-0.0196 µg mL-1, respectively. This method is not only applicable to routine analysis of SPFX in bulk and pharmaceutical formulations but as well as on human serum samples. Key words: Sparfloxacin, reverse phase high-performance liquid chromatography (RP-HPLC), serum, analysis of variance (ANOVA), student’s t-test.