The search for new potent anticancer drugs that can only target cancer cells, rather than affecting normal tissues is very much commendable. Supramolecular Calixarene (p-tert-butylcalix[4]arene) is a highly promising candidate in this regard and could be modified to enhance preferential cytotoxicity for targeted therapy. Calixarenes are a family of bowl or cone shaped synthetic supramolecular macrocycles, composed of phenol units linked by methylene bridges through and an aldehyde. The proliferation rate of L-929 were observed higher than MCF-7 cells in the Real-Time Cell Analysis (RTCA) Systems. Our research directs that calixarene shows effective preferential cytotoxicity at a concentration range of 25 to 100 μg/mL with enhanced preferential cytotoxicity shown by the modification of pyridinium group at the position 3 against MCF-7 breast cancer cells. In this present study, the effect of surface altered calixarene by pyridinium groups on preferential cytotoxicity in cancer cell in-vitro by comparing with innate preferential toxicity shown by unaltered. The results reported in this study demonstrated that tumor-preferential in-vitro cytotoxicity of p-tert-butylcalix[4]arene against MCF-7 over L-929 cells present a promising approach for efficient and safe cancer therapy.