Gastric cancer (GC) is an aggressive malignancy with increased mortality rate and low treatment options. Increasing evidence suggests that network pharmacology will be a novel method for identifying the systemic mechanism of therapeutic compounds in diseases like cancer. The current study aimed to use a network pharmacology approach to establish the predictive targets of prunetin-5-O-glucoside (PG) against gastric cancer and elucidate its biological mechanisms. Primarily, genes associated with the pathogenesis of GC was identified from the DiGeNET database and targets of PG was obtained from the Swiss target prediction database. In total, 65 correlative hits were identified as anti-gastric cancer targets of PG. Functional enrichment and pathway analysis revealed significant biological mechanisms of the targets. Interaction of protein network and cluster analysis using STRING resulted in three crucial interacting hub targets namely, HSP90AA1, CDK2, and MMP1. Additionally, the in vitro cytotoxic potential of PG was assessed on three gastric cancer cells (AGS, MKN-28, and SNU-484). Furthermore, the crucial targets were validated using molecular docking, followed by their expressions being evaluated by western blot and Human Protein Atlas. The findings indicate that the pharmacological action of PG against GC might be associated with the regulation of three core targets: HSP90AA1, CDK2, and MMP1. Thus, the network pharmacology undertaken in the current study established the core active targets of PG, which may be extensively applied with further validations for treatment in GC.