To detect the amount of rifampicin in bulk and medicinal dosage formulations, an accurate and costeffective UV spectrophotometric technique has been developed using the area under the peak to estimate the presence of rifampicin. This range of wavelengths (300–356 nm) was chosen. The method showed linearity in the 2–22 μg/mL range, with R2 being2 0.9996. The developed method’s linearity, detection limit, quantification limit, precision, repeatability, and accuracy were all statistically and experimentally validated. The suggested methodology can be used for routine quality control analysis of rifampicin in pure form and in capsule dosage form, as demonstrated by the satisfactory recovery percentage results. This study explores the structural and electronic properties of rifampicin using density functional theory (DFT) and its interaction with potential biological targets via molecular docking. The DFT analysis, conducted using the B3LYP functional and a suitable basis set, provides detailed insights into the optimized molecular geometry, frontier molecular orbitals (FMOs), and molecular electrostatic potential (MEP) of rifampicin. The energy gap (ΔE = 2.878 eV) exported the stability conditions of rifampicin. Reduced density gradient analysis (RDG/NCI) was considered to highlight the specific interactions present inside the molecule, predicting its stability. Molecular docking studies complement the DFT analysis by identifying rifampicin binding affinity (-36.01 kcal/ mol) with the specific 5F92 target protein. This study evaluates the ADMET properties of Rifampicin to assess its pharmacokinetic and safety profile.