Critical cytotoxicity evaluation of pharmaceuticals is necessary for the clinical practice of chemotherapy. To quantitatively evaluate cell viability, currently there are two main types of sensitive methods including real-time cell analysis (RTCA) and CCK-8 assay, in which RTCA records electrochemical signal changes around an incubated cell, whereas CCK-8 is based on the colorimetric method. Despite the different detection principles adopted for the cytotoxicity assessment, the comparison of the two methods in terms of the application scope is lacking. In this study, comparison studies were conducted between the RTCA and CCK-8 assays using anticancer drugs including doxorubicin hydrochloride, curcumin, irinotecan (CPT-11), taxol, and oxaliplatin, which are classified into two groups of drug molecules in the absence and presence of additives. The cytotoxicity evaluation of these drugs on cancer cells revealed that the physicochemical properties of drug formulations such as optical and electrochemical properties are closely linked with the readout of cytotoxic methods. The experimental results suggested that the preselection of cytotoxic assay is critical for the quantitative measurement of cytotoxicity of anticancer drugs, which is of clinical importance for their therapeutic usage.