Purpose: To establish the method for the analysis of rifampin quinone and hydrazone in fixeddose combination tablets (FDC) containing rifampicin with or without sodium ascorbate as an additive. Methods: The International Pharmacopoeial (IP) method for the estimation of rifampin quinone in FDC was modified. The extraction solvent (methanol/buffer mixture) of the IP method was replaced with ethyl acetate, in order to stabilize rifampin quinone in sample solutions of FDC containing sodium ascorbate as an ingredient. Results: Rifampin quinone in FDC samples containing sodium ascorbate, which is not detectable in the IP method, was found to be 1.15 % (4-FDC), 1.52 % (3-FDC) and 1.60 % (2-FDC) using the modified method. Recovery of rifampin quinone in spiked samples of FDCs was practically nil in IP method whereas the recovery was 99.95%, 99.39 and 99.02 % for 4–FDC, 3-FDC and 2-FDC, respectively. Conclusion: This modified method was suitable for the determination of rifampin quinone in fixed-dose formulations of rifampin both in the presence and absence of sodium ascorbate. The method is specific, precise, accurate, robust, rugged and gives a linear response for the quantitative estimation of rifampin quinone and hydrazone in fixed-dose combination tablets containing rifampin.