The cytotoxicity test is one of the biological evaluation and screening tests that use tissue cells in vitro to observe the cell growth, reproduction and morphological effects by medical devices. Cytotoxicity is preferred as a pilot project test and an important indicator for toxicity evaluation of medical devices as it is simple, fast, has a high sensitivity and can save animals from toxicity. Three types of cytotoxicity test are stated in the International Organization for Standardization 109993-5: Extract, direct contact and indirect contact tests. The xCELLigence real-time cell analysis system shows a significant potential in regards to cytotoxicity in recent years. The proliferation of L-929 were observed in the Real-Time Cell Analysis (RTCA) Systems. Our research directs that calixarene shows no cytotoxicity at a concentration range of 25 to 100 μg/mL on L929 cells. There were no significant differences between the L929 cell indexes of the control and all testing groups at 24, 48 and 72 h.