A novel, selective, precise, and accurate RP-HPLC method was developed and validated for simultaneous estimation of Etoposide and Picroside-II in bulk and the patented pharmaceutical formulation. The chromatographic analysis was performed on Agilent 1260 Infinity HPLC instrument using a Kromasil C-8 column (150 × 4.6 mm, particle size-5 um) and a mobile phase comprising 0.5% acetic acid in water: actonitrile (75:25 v/v). Flow rate of mobile phase was kept at 0.8 ml/min and the column eluent was monitored at 254 nm. The total run time was 15 min and the average retention time of Etoposide and Picroside-II was found to be 13.68 min and 3.62 min respectively. The calibration curves of Etoposide and Picroside-II were linear over the range of 1-10 ng/mL with R2 = 0.999. Average percentage recoveries of Etoposide and Picroside-II were 100.27±0.49 and 100.39±0.23 % respectively. The LOQ values of Etoposide and Picroside-II were 7.52 and 9.11 ng/mL respectively. Proposed method was found to be precise and reproducible as the RSD values of intra-day and inter-day precision studies were below 2%. Proposed method was successfully used for the quantitative analysis of Etoposide and Picroside-II in patented pharmaceutical formulation wherein the assay content of Etoposide and Picroside-II was found to be 100.27 and 99.92% respectively.