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A simple and sensitive reversed phase HPLC method has been developed for the simultaneous quantitation of pretomanid (PA-824), moxifloxacin and pyrazinamide in a combination spray-dried powder formulation for inhalation, without any use of an internal standard. Good resolution of the analytes was achieved on a Luna C18 (2), 150 × 4.6 mm, 5 μm, 100 Å column using gradient elution with a mobile phase containing methanol and triethylamine phosphate buffer (pH 2.5) at a flow rate of 1.0 mL/min in a total run time of 25 min. Pyrazinamide, moxifloxacin and pretomanid (PA-824) were detected at wavelengths (retention times) of 269 nm (3.80 min), 296 nm (7.94 min) and 330 nm (17.46 min), respectively. The assay was linear for all analytes in the concentration range 2.5–100 μg/mL (correlation coefficients >0.999) with LODs and LLOQs (μg/mL) of pretomanid (PA-824) 0.51 and 1.56, moxifloxacin 0.06 and 0.19 and pyrazinamide 0.35 and 1.06, respectively. Recoveries of the three drugs were 99.6–106.8% with intra- and inter-day precisions (as relative standard deviation) of <7%. The method was successfully applied to an evaluation of content uniformity and freedom from interference by L-leucine of a spray-dried combination powder for inhalation.Development of a RP-HPLC method for the simultaneous quantitation of pretomanid (PA-824), moxifloxacin and pyrazinamide.Simple, accurate, precise and sensitive method to analyze three drugs.Freedom from interference by L-leucine.Successful evaluation of content uniformity of a triple combination spray-dried powder.