CPZEN-45 is a new drug candidate being considered for the treatment of tuberculosis (TB). The aim of this study was to develop and validate a reverse-phase high-performance liquid chromatographic (HPLC) method suitable to determine CPZEN-45 concentrations in biological samples. CPZEN-45 was extracted from biological fluids and tissues (plasma, lung and spleen from guinea pig) by sequential extraction with acetonitrile and quantified by a Waters HPLC Alliance System coupled with a ZORBAX Bonus-RP column, guard column and UV detection at 263 nm. The mobile phase was 20:80 acetonitrile:ultrapure-water with 0.05% TFA. The CPZEN-45 peak was eluted at 5.1 min with no interference from the inherent peaks of plasma, bronchoalveolar lavage (BAL), lung or spleen tissues. Recovery of CPZEN-45 from biological samples was >96% of the spiked amount. The limit of detection was 0.05 µg/ml and the limit of quantitation was 0.29 µg/ml which was more than 5 and 21 times lower than the reported minimal inhibitory concentration of CPZEN-45 (MIC = 1.56 µg/ml for Mycobacterium tuberculosis and 6.25 µg/ml for MDR-TB, respectively). Thus, HPLC method was deemed reliable, sensitive, reproducible and accurate for the determination of CPZEN-45 concentrations in plasma, BAL, lung and spleen tissues. Therefore, this method was used in subsequent studies in the guinea pig model to determine the disposition of CPZEN-45 after administration of solutions by the IV and SC routes.