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The plant Nymphaea pubescens Willd. (Family: Nymphaeaceae) is edible having medicinal importance. The objective of the study was to analyze the potential hepatoprotective properties of the flowers and pedicels of N. pubescens by inhibiting the enzyme β-glucuronidase. Crude methanol extracts of flower and pedicel as well as chloroform, ethyl acetate, and aqueous fractions of the flower extract were tested for their activities against the enzyme in vitro. The extracts and the fractions were analyzed by GC–MS to identify metabolites present in them. Flower (IC50 value = 270.27 ± 4.67 μg/ml) and pedicel (IC50 value = 868.46 ± 28.21 μg/ml) extracts have shown to inhibit the β-glucuronidase activity. Chloroform (IC50 value = 147.16 ± 6.68 μg/ml), ethyl acetate (IC50 value = 183.94 ± 2.37 μg/ml), and aqueous (IC50 value = 339.43 ± 5.34 μg/ml) fractions showed significantly stronger activity than that of silymarin (IC50 value = 792.62 ± 10.01 μg/ml), the known inhibitor of the enzyme. GC–MS-based analysis of the flower extract and solvent fractions led to the identification of kaempferol having 79-fold stronger activity than that of silymarin, IC50 value of kaempferol being 10.44 ± 0.084 μg/ml or 0.0037 mM ± 0.0001.