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According to ICH Q2 (R1) criteria, two different, simple and sensitive densitometric High Performance Thin Layer Chromatography methodologies for assessment of Berberine HCl (BER) and Thymoquinone (TQ) in bulk individually and in established microemulgel formulation has been developed and validated. The analysis was conducted separately for BER and TQ on Merck HPTLC aluminum sheets of silica gel 60 F254 by using a mixture of Ethanol: Glacial acetic acid: Water (16:2:2 v/v/v) for BER and Toluene: Acetone: Formic acid (5:4:0.2 v/v/v) for TQ as a mobile phase and densitometric determination was carried out by TLC scanner (CAMAG) at 366 nm and 254 nm respectively for BER and TQ. The approach produced well-defined peaks for BER and TQ with Rf values of 0.468 and 0.817, respectively. The calibration plots for BER and TQ exhibited a satisfactory linear relationship with regression coefficient (r2) of 0.9991 and 0.9953, respectively, in the range of concentrations of 50-350 ng/band and 50-300 ng/band. The limit of detection and quantification were 10.76 and 32.65 ng/band respectively for BER, 3.77 and 11.43 ng/band respectively for TQ. Further, the study provides additional evidence that accuracy, precision and repeatability were all within the acceptable range. The method has been successfully applied to the identification and quantitative determination of BER and TQ in bulk and its combination containing formulation.