In the present work, highly porous, pH-responsive, and biocompatible chitosan-based hydrogel beads were prepared through gamma-irradiated graft copolymerization technique using L-glutamic acid as the monomer. The glutamic acid grafted chitosan (CH-g-GA) hydrogel beads, loaded with the anti-cancer drug (Doxorubicin, Dox), were exploited for their potential application as anti-cancer drug delivery system. The grafting conditions were optimized by varying irradiation dose (kGy) and monomer concentration. Further, the hydrogel beads were analysed using FTIR, XRD, SEM, TGA/DSC, Zeta potential studies, BET analysis and their strength was determined using rheological analysis. The swelling characteristics of the beads were studied at various simulated body pH (2.1, 5.8, and 7.4) to study their pH-responsive behaviour. The in-vitro drug release from the beads was thus evaluated at pH 5.8, 7.4 using UV-visible spectroscopy. The highest swelling ratio (426%) and drug release (81.33% in 144 h) was observed at the pH of 5.8. The MTT assay was performed on HEK-293 cell-line to check their cytocompatibilty and the cell proliferation of Dox-loaded beads was studied on MCF-7 cell-line. A significant cytotoxicity against the cancer-cells was observed which further established their promising use in the controlled delivery of anti-cancer agents for localized cancer therapy.