There is increasing interest in the rapid and highly sensitive monitoring of cell viability in biological and toxicological research. Conventionalmethods depend on optical assays using Water Soluble Tetrazolium-8 (WST-8) or 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) assay, which requires a large volume of samples and special instruments, necessitating shipment ofclinical samples to laboratories. This paper reports on the development of a rapid and sensitive electrochemical (EC) sensor using screenprinted electrode (SPE) and surface modification using 4’-mercapto-N-phenylquinone diamine (4’-NPQD), as double electron mediators,for monitoring cell viability via the measurement of nicotinamide adenine dinucleotide (NADH). We used the sensor to observethe viability of MCF-7 and doxorubicin (Dox)-treated cells. The oxidation current of NADH was measured via chronoamperometry(CA), and the EC results showed a good linear relationship when compared with NADH quantification using WST-8 assay. The analysistime was only 10 s and limit of detection (LOD) of NADH was 1.78 μM. Our EC method has the potential to replace conventionalWST assays for cell viability and cytotoxicity experiments.