Tuberculosis (TB) remains a life-threatening infection, and it is well-known that effective TB treatment is associated with multiple drugs administered to infected patients on a daily basis. Terizidone (TZD) is an anti-TB drug used in the treatment of multi-drug resistant and extensively drug-resistant TB but presents with polyneuropathic adverse effects in some patients. To counteract these adverse effects, TZD is typically prescribed with pyridoxine (PDX), well known as Vitamin B6. As part of a pre-formulation study investigating the potential to co-formulate these two compounds, it became necessary to have a simple and reliable reversed-phase high-performance liquid chromatography (RP-HPLC) method. Optimal, simultaneous separation and detection of TZD and PDX were obtained using an isocratic mobile phase setup, consisting of ultrapure water and acetonitrile (30:70% v/v), with 1 mL glacial acetic acid added to the mobile phase mixture. A Discovery® C18, 150 × 4.6 mm, 5 μm column maintained at ambient temperature was utilized, with a detection wavelength of 260 nm. The method was validated in terms of linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), specificity, robustness, and solution stability. Validation proved this method to be acceptable and reliable for the simultaneous accurate detection and quantification of TZD and PDX.