This report describes a fast, simple and reliable HPLC method for the assay and quantitative determination of calcium pantothenate in commercial products. The samples were analyzed on a C18 column with the mobile phase of acetonitrile and potassium dihydrogen phosphate solution (adjusted pH = 2.5 with phosphoric acid) at a flow rate of 1.0 mL/min and UV absorbance detection at 204 nm. Ampicillin was used as an internal standard. The retention times of calcium pantothenate and ampicillin were 5.3 and 6.5 min, respectively. An equation was presented for linear relationship between peak height ratios of calcium pantothenate to ampicillin and the calcium pantothenate concentration over a range of 10-50 μg/mL (r = 0.9999). Standard addition recoveries were greater than 98.96% with twelve commercial products. The relative standard deviations were between 0.1 and 0.9% in inter-day assays, 0.1 and 0.7% in intra-day assays. The results obtained from the HPLC assay method which we developed and the microbiological assay of USP were paired at 95% confidence level. There were no significant differences between these two methods. The proposed HPLC method was a suitable substitute for microbiological method for quantitative assays of calcium pantothenate in commercial products.