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Asensitive, selective, and precise high-performance thin-layer chromatography (HPTLC) method was developed for simultaneous analysis of six bioactive phenolic compounds, i.e., juglone, quercetin, myricetin, rutin, caffeic acid, and gallic acid in methanol extract and its fractions (hexane, chloroform, ethyl acetate, and butanol fractions) from bark of Juglans regia. Good separation was achieved on RP-18 F254S TLC plate using methanol-water-formic acid-acetic acid (48.8:46.4:2.4:2.4, ν/ν). The densitometric determination of the compounds was carried out at 254 nm in reflectance/absorbance mode. The method was validated in terms of linearity, sensitivity, accuracy, precision, robustness, and specificity. The linear regression data for the calibration plots of the reference compounds showed a good linear relationship with higher correlation coefficient (r 2 ≥ 0.997). Accuracy of the method was evaluated in terms of average percent recovery, which ranged from 98.63 to 101.06%. HPTLC results revealed qualitative and quantitative differences in the phenolic compounds in the extract and fractions. The ethyl acetate fraction contained gallic acid followed by myricetin, rutin, quercetin, and caffeic acid in higher amount in comparison to the extract and fractions. The present method can be used for routine quality control of J. regia extracts.

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