Tuberculosis could be regarded as a serious and enduring menace to global health. A significant hurdle in tuberculosis treatment lies in drug resistance, including the emergence of multiple drug resistance. A regulatory agency has recently endorsed the fixed-dose combination of pyridoxine HCl, isoniazid, trimethoprim, and sulfamethoxazole as a robust therapy for cases of tuberculosis resistant to multiple drugs. Therefore, a successful attempt was made to develop and validate a novel spectroscopic method, i.e., double deviation ratio spectra derivative spectroscopic method, for the simultaneous determination of pyridoxine HCl, Isoniazid, trimethoprim, and sulfamethoxazole in their pure form. The optimized divisor concentration in the developed method was found to be a mixture of 6 μg/mL of sulfamethoxazole and 16 μg/mL of pyridoxine HCl for the determination of isoniazid and trimethoprim in the same quaternary mixture. Similarly, the optimized divisor concentration for the determination of sulfamethoxazole and pyridoxine HCl was found to be a mixture of isoniazid and trimethoprim with concentrations of 10 and 12 μg/mL, respectively. With the developed method, the linearity for pyridoxine HCl, isoniazid, trimethoprim, and sulfamethoxazole was found to be in the range of 8 to 40 μg/mL, 10 to 30 μg/mL, 12 to 36 μg/mL, and 4 to 12 μg/mL, respectively. The results of the accuracy study, in terms of percentage recovery, were found to be 99.92 ± 0.978, 100.04 ± 1.731, 99.87 ± 1.811, and 98.83 ± 0.922 for the simultaneous determination of pyridoxine HCl, isoniazid, trimethoprim, and sulfamethoxazole, respectively, using the developed method. The developed method has been successfully validated as per the ICH guidelines.