Bedaquiline, a highly lipophilic molecule, is used in the treatment regimen of multi-drug resistant tuberculosis. A rare complication of pulmonary tuberculosis is tuberculous pericarditis. Ex vivo studies utilizing animal pericardium can be used to investigate whether this drug is capable of diffusing across pericardial tissue into simulated pericardial fluid (pH 7.4) to indicate efficacy. For detection of bedaquiline in physiological fluid, a rapid, cost-effective and sensitive method is essential. The aim of this study was thus to develop and validate a simple and sensitive RP-HPLC-UV method for the detection and quantification of bedaquiline, encapsulated in a nanosystem, at pH 7.4 after permeation across excised pericardium. A HPLC Phenomenex Kinetex RPC18 column (150 x 4.6 mm, 5 µm) was utilized for analysis. The mobile phase consisted of 95:5 v/v (A: B), where (A) Methanol: Acetonitrile (85:15 v/v): (B) Triethylamine (1% v/v):0.15 mM KH2PO4 buffer (pH 7.4). Running conditions included the following: injection volume 20 µl, flow rate 1.0 ml.min-1, detection wavelength 275 nm, 25oC and running time of 5 min. Bedaquiline eluted as a single symmetrical peak at a retention time of 4.17 min. The method was found to be linear within the range of 1 - 50 μg.ml-1 (R2 = 1). The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.05 μg.ml-1 and 0.15 μg.ml-1, respectively (signal-to-noise ratio method). All validation parameters were found to be within acceptable limits (RSD<2%). The method was fast, reliable, accurate, reproducible, and transient for the detection of bedaquiline in simulated physiological fluid (pH 7.4). This method can thus be applied to easily detect bedaquiline in body fluids (pH 7.4) i.e. blood and pericardial fluid without the accuracy being impacted by ionization factors of the molecule.