The method was validated via calculating linearity, peak purity, limit of detection (LOD), limit of quantification (LOQ), repeatability, percentage recovery, intra-day and intermediate precision.Each of the standard solutions of barbaloin (0.3, 0.6, 0.9, 1.2, 1.5 μg per band) was administered in triplicate. The calibration plot was developed by plotting peak area against the amount of barbaloin and linearity range was resolved.