BACKGROUND: The association between Tuberculosis (TB) and Diabetes Mellitus (DM) have always been an area of interest for decades. Many studies have reported the impact of DM on the development and prognosis of TB and vice versa. There is a major research gap in the area of drug-drug interactions between anti-TB drugs and anti-diabetic drugs. Hence, this study investigated the drug-drug interactions between first-line anti-TB and anti-diabetic drugs in patients with TB and DM. METHODOLOGY: A prospective study was undertaken in three groups of adult patients, namely, TB, DM and DMTB from May 2018 to February 2020. Plasma concentrations of RMP, INH, PZA, MET, GLB, GLP and GLM were estimated by high-performance liquid chromatography (HPLC). RESULT: 21 TB, 76 DMTB and 51 DM patients with subgroups in both DM and DMTB were recruited. It was observed that plasma levels of anti-diabetic drugs were altered in DMTB patients receiving first-line anti-TB drugs. However, the levels of anti-TB drugs were not altered in the presence of DM and irrespective of the DM status, sub-therapeutic rifampicin concentration was detected. CONCLUSION: This study provided the roadmap to study the impact of anti-diabetic drug interactions on anti-TB drugs and vice versa. Our study is the first of its kind to explore this puzzle.