Abstract The study focused on the simultaneous quantification of Isoniazid (INH) and Pyridoxine Hydrochloride (PRD) in tablet form commonly used in antituberculosis treatments. Assessing the accurate concentration of both INH and PRD in tablets is vital to ensure their effectiveness safety and quality. Using the ratio subtraction spectrophotometry method the study analyzed INH and PRD in Pehadoxin forte reg tablets (batch no. 36057007 PT. Phapros Indonesia) with 0.1 N HCl as the solvent. The method involved obtaining ratio absorption spectra by dividing the absorption spectra of INH and PRD respectively to derive zero-order spectra for each drug. Method validation parameters included linearity accuracy precision Limit of Detection (LOD) and Limit of Quantification (LOQ). The results showed linearity values of 0.9985 for INH and 0.9988 for PRD. Accuracy was 98.1838% for INH and 100.0205% for PRD while precision was 1.8769% for INH and 0.2037% for PRD. LOD and LOQ for INH were 0.8116 micro g/mL and 2.7053 micro g/mL respectively and for PRD 1.3127 micro g/mL and 4.3757 micro g/mL. The levels of INH and PRD in the tablets were found to be 102.1157% and 101.3874% aligning with the Indonesian Pharmacopoeia s standards. This methodological approach provides a reliable analytical tool for the simultaneous assessment of INH and PRD in tablets potentially extendable to other drug combinations and formulations thereby contributing to pharmaceutical quality control processes. Keywords isoniazid pyridoxine hydrochloride ratio subtraction spectrophotometry