A precise, simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed and validated for qualitative and quantitative estimation of lincomycin hydrochloride in combined drug substance and in sterile powder for oral solution. Chromatographic separation was performed by Shimadzu Prominence-i LC 2030 C with ZORBAX SB-C18 column (3.5µm, 4.6 mm×75 mm)at ambient temperature with isocratic mobile phase consisting of 67:33 (v/v) mixture of 0.023 M orthophosphoric acid (pH adjusted to 2.3) and acetonitril as mobile phase, at a flow rate of 1.0 ml/min with UV detection at 210 nm. The method was validated for linearity and range, accuracy, specificity, robustness, precision (intra and inter-day) and limit of quantification and limit of detection. The calibration plot was linear from 5.0 -100 µg/ml with regression equations y = 33.605x + 5.6529 and correlation coefficient (R²) of 0.9999. In conclusion, the developed method was precise, simple and rapid for estimation of lincomycin hydrochloride.