A simple, precise and reliable RP-HPLC (Reverse phase high-performance liquid chromatography method) was developed and validated for the simultaneous estimation of calcium pantothenate (CP) and biotin (BI) in pure and their tablet dosage form. The chromatographic separation is being achieved by using Cosmosil C18 column (250 mm × 4.6 mm i.d, 5 µm) and mobile phase consisting of buffer (Mono basic potassium dihydrogen orthophosphate with 10% orthophosphoric acid, pH adjusted to 2.20 ± 0.05) and methanol (70:30% v/v) with the flow rate of 1.0 ml/min. The PDA detector was used as detector, measuring the response at 210 nm. The retention time was found to be 4.5 and 9.8 min for CP and BI respectively. The linearity of the drugs was acquired in the range of 50-150 µg/ml and 5- 15 µg/ml for CP and BI respectively. ICH guidelines were used for the validation of the results obtained.