B Background: Plectranthus amboinicus is a plant that has been used by the Batak tribe as traditional medicine. Research shows that the extracts of n-hexane, ethylacetate and ethanol from P.amboinicus. has antioxidant activity. Antioxidant activity is usually correlated with cancer prevention. Purpose: This study was designed to determine the effect of P.amboinicus tuber ethanol extract on the cytotoxicity of HSC-3 cell lines. Method: HSC-3 cell lines were treated with ethanol extract of P.amboinicus tubers with a concentration of 100 ppm, 600 ppm and 800 ppm for 24 hours, then tested with MTT to determine optical density and determine cell inhibition followed by probit analysis to find IC50 to determine the effect of cytotoxicity. Results: MTT test results showed P.amboinicus leaf extract fraction of n-hexane, ethyl acetate and ethanol had an effect on cell count and apoptosis induction in HSC-3 cell lines. The treatment of P.amboinicus leaf extract with n-hexane fraction with a concentration of 100 ppm, 600 ppm and 800 ppm can inhibit cell growth by 19%, 36% and 34%, the ethanol fraction with a concentration of 100 ppm, 600 ppm and 800 ppm can inhibit growth cells of 30%, 14%, and 10%, while the ethanol fraction at concentrations of 100 ppm, 600 ppm and 800 ppm had inhibition of 37%, 11% and 16%. The results of the MTT test resulted in the IC50 value in the n-hexane fraction with a concentration of 430.622 ppm, the ethyl acetate fraction of 56.098 ppm and the ethanol fraction of 149.575 ppm. The results of statistical analysis with ANOVA on the n-hexane fraction showed (p = 0.030) ethyl acetate fraction (p = 0.320) and the Kruskal-Wallis test on ethanol fraction (p = 0.032). Conclusion: P.amboinicus leaf extract fraction of n-hexane, ethyl acetate and ethanol had a cytotoxic effect on HSC-3 cell lines.