A highly sensitive and selective LC-MS/MS assay was developed and validated for determination of a novel antitubercular compound S006-830 in rat plasma. The analyte and internal standard (IS) were extracted from plasma by a two step liquid–liquid extraction procedure using 2% isopropanol in n-hexane. Chromatographic separation was achieved on a Phenomenex, Luna C-18 column (3µm, 100mm x 2mm i.d.) under isocratic condition [92:8 (v/v); ACN (0.1% formic acid) : ammonium acetate buffer (10 mM, pH 4)] at a flow rate of 0.450 ml/min. The quantification was performed on Qtrap 5500 LC-MS/MS coupled with ekspert ultra LC 100-XL system (AB Sciex). The detection was performed in positive electrospray mode using multiple reaction monitoring. The precursor to production of ion transitions selected for quantification of S006-830 and IS was m/z 424.353/203.00 and 330.300/267.400 respectively. LC-MS/MS method was found sensitive and reproducible over a linearity range of 0.15-40 ng/ml. Recoveries of S006-830 from spiked plasma samples were consistent and found to be more than 70%. Further, the applicability of this method has been described by determining pharmacokinetic (PK) profile and plasma protein binding of S006-830 in rats. Irregular plasma-concentration time profile was observed. Oral PK profile of S006-830 at 50 mg/Kg demonstrated that mean (±SEM) T1/2 and mean residence time were 8.30 ± 1.30 h and 8.44 ± 0.57 h, while Cmax and AUC0-last were 1.94 ± 0.30 µg /ml and 6.25± 1.66 μg.h /ml respectively. Plasma protein binding for S006-830 was found to be 58.63 ± 3.4 %.