Abstract: Background and Objective: Chillies are delicious spices that are used extensively. Capsaicinoids, the major constituents of chillies with reported anti-cancer effects, have been determined with non-specific colorimetric methods. A rapid and reproducible method for extraction and quantification of the major chillies capsaicinoids; capsaicin, dihydrocapsaicin (DHC) and nordihydrocapsaicin (n-DHC), was reported, moreover study of their cytotoxic activity. Materials and Methods: This study has covered the extraction of capsaicinoids from red and green-colored chillies followed by their quantification using HPLC-UV method after validation. Furthermore, the correlation of capsaicinoids contents with their in vitro hepatocarcinoma cytotoxicity was represented by Pearson’s correlation coefficient. Results: Capsaicinoids contents are ranged from 1219.88-15098.67 ng mg–1 of Dried Extract (DE). Capsaicin exhibits the lowest IC50 when compared to doxorubicin (9.201±0.91 and 16.1±0.82 μg mL–1, respectively). The exhibited activities of methanol extracts of red and green-colored chillies (IC50 = 20.21±1.72 and 16.02±0.69 μg mL–1, respectively) may attribute to their excessive contents of capsaicinoids (6975.42 and 15098.67 ng mg–1 DE, respectively). Capsaicin and n-DHC contents have a negative correlation with cytotoxic activity. Conclusion: Green-colored chillies were found to be more cytotoxic in comparison with red-colored chillies that may be relative to their high content of capsaicinoids. The present investigation suggests that capsaicinoids contents correlate with cytotoxic activity.