IDP-73152, a novel inhibitor of a bacterial peptide deformylase, was recently approved as a new, investigational drug in Korea for the clinical management of infections caused by Gram positive bacteria. The objective of this study was to develop/validate a simple and robust analytical method for the determination of IDP-73152 in plasma samples from rodents and humans, and to assess the feasibility of the assay for use in pharmacokinetic studies using animal models. Plasma samples were processed using a standard method for protein precipitation and an aliquot of the extract then injected onto an UHPLC–MS/MS system. The drug and IDP-117293, an internal standard, were analyzed in the positive ion-mode by electrospray ionization and quantified by monitoring the transition at m/z 555.2 → 245.2 for IDP-73152 and 563.3 → 253.1 for the internal standard, respectively. The lower and upper limit of the assay was determined to be 5 and 10000 ng/ml, respectively, with an acceptable linearity (R > 0.999) in the response-concentration relationship. Validation parameters, including accuracy, precision, dilution, recovery, matrix effect and stability were found to be within the acceptable ranges recommended by the assay validation guidelines of the United States FDA. The method was successfully applied to the quantification of IDP-73152 in plasma from mice/rats that had received a single oral administration of 80 mg/kg IDP-73152, in the form of the mesylate salt. These findings suggest that the validated assay can be used in preclinical and clinical pharmacokinetic studies of IDP-73152.