Abstract: Glycyrriza glabra Linn.[ Fabaceae], commonly known as yashtimadhu, mulhatti, is used as medicine for treatment of various aliments in Traditional System of Medicine fairly throughout the greater part of India. In the present study, we have evaluated anticancer activity of Ammonium glycyrrhizinate by in vitro and in silico method. Ammonium glycyrrhizinate was isolated from roots of G.glabra and was characterized by solubility, melting point, infrared (IR) and thin layer chromotography (TLC) pattern. In vitro anticancer activity was done using HeLa cell lines by MTT assay at different concentrations ranging from 100-500 µg/ml using microtitre plate assays by ELISA and in silico docking studies using enzyme EGFR tyrosine kinase. The IC50 value was found to be 282.45 µg/ml in in vitro anticancer activity in HeLa cell lines. Ammonium glycyrrhizinate was subjected to molecular docking studies for the inhibition of the enzyme EGFR tyrosine kinase, which is one of the targets for inhibition of cancer cells. It has shown -11.03 kJ mol-1 binding and -12.47 kJ mol-1 docking energy with five hydrogen bonds. We can conclude that ammonium glycyrrhizinate has shown to possess anticancer activity both in vitro and in silico studies.