A simple, rapid, and specific reversed-phase HPLC method has been developed for simultaneous analysis of withaferin-A and 6-gingerol in a polyherbal formulation containing Withania somnifera and Zingiber officinalis extracts. HPLC analysis was performed on a C18 column using a 40:60 (v/v) mixture of acetonitrile and water as isocratic mobile phase at a flow rate of 1.5 mL min−1. UV detection was at 227 nm for withaferin-A and 278 nm for 6-gingerol. The method was validated for accuracy, precision, linearity, specificity, and sensitivity in accordance with International Conference on Harmonization guidelines. Validation revealed the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r2 > 0.9996) were obtained for calibration plots in the ranges tested. Limits of detection were 0.2 and 0.4 μg and limits of quantification were 0.5 and 1.0 μg for withaferin-A and 6-gingerol, respectively. Intra and inter-day RSD of retention times and peak areas were less than 2.1%. Recovery was between 94.5 and 98.8% for withaferin-A and 94.2 and 102.4% for 6-gingerol. The established HPLC method is appropriate and the two markers are well resolved, enabling efficient quantitative analysis of withaferin-A and 6-gingerol. The method was successfully used for quantitative analysis of these two marker constituents in a marketed polyherbal formulation.