In this study, several novel β-carboline derivatives, 1-(4-hydroxy-3-methoxyphenyl)-β-carboline-3-carboxyl-Trp-Trp-AA-OBzl compounds, were designed and synthesized as potential anticancer agents. Their in vitro cytotoxic activities were evaluated using methylthiazoltetrazolium (MTT) assay. The in vivo anti-tumor activity of the newly synthesized β-carboline derivatives was determined in a S180 bearing mouse model and some of the compounds demonstrated tumor growth inhibition similar to the positive control, doxorubicin. The intercalation of the β-carboline derivatives synthesized with calf thymus (CT) DNA was also studied.