Abstract: Background and Objective: Annona reticulata or custard apple belongs to family Annonaceae. It is traditionally used to treat various human ailments. However, there are no studies on the cytotoxicity or apoptosis-inducing properties of the leaf extracts on HT-29 cell line. Hence, the present study aimed at screening the anti-cancer potential of Annona reticulata leaf extract through various in vitro studies. Materials and Methods: The methanolic leaf extract of Annona reticulata (ARM) was subjected to quantification of flavonoids by UPLC/MS; further analyzed for its cytotoxic effect by MTT assay, cell cycle analysis, apoptotic potential by Annexin V-FITC staining assay and morphological study by phase contrast microscopy. Results: The ARM showed significant dose-dependent cytotoxicity towards HT-29 cell lines with IC50 of 76.76 μg mL–1. In cell cycle analysis, ARM 160 μg mL–1 exhibited significant (p<0.001) increase in the percentage of cells at S phase, indicating the induction of apoptosis. Further, apoptosis induction was confirmed by Annexin V-FITC assay and morphological evaluation. The results showed that the percentage of late apoptotic cells were found to be higher in ARM 160 μg mL–1 treated cells (82.53%) compared to untreated (0.71%) cells. Also, ARM 160 μg mL–1 showed similar activity as colchicine treated cells (82.18%). Under morphological evaluation, the formation of apoptotic bodies was found to be more evident in ARM compared to colchicine. Conclusion: These findings suggest ARM as a potent anti-cancer agent and also provide a basis for further studies validating ARM as an adjuvant in cancer therapy.