Cell proliferation assays are basic and essential techniques for assessing cellular function. Various colorimetric assays, such as MTT-, WST-1-, and resazurin-based assays, are available; however, studies directly comparing the suitability of each method for immune cell proliferation are scarce. Thus, we aimed to determine the best reagent and its optimal conditions based on variables such as cell number range, stimulation dose, kinetics, and compatibility with the cell division assay using CFSE fluorescence dye which is able to directly monitor divided cells by flow cytometry. In the absence of stimulation, MTT solubilized with SDS (MTT-SDS) and resazurin appeared to accurately reflect the cell numbers in a linear fashion. On the other hand, WST-1 exhibited a higher stimulation index following strong stimulation, whereas MTT-SDS and resazurin exhibited a better sensitivity to weak stimulation. A longer duration for stimulation did not necessarily increase sensitivity. CFSE staining revealed incremental cell division in response to anti-CD3 antibody stimulation in a dose-dependent manner. The cell numbers indirectly estimated from cell division profiles were consistent with the dose-response curve in the absorbance of MTT-SDS and resazurin. The absorbance does not increase before cell division, irrespective of T cell activation status, suggesting that these reagents reflect the cell number but not the cellular volume. Collectively, resazurin and MTT-SDS seem to be more reliable than others, and thus appear applicable in various conditions for the immune cell experiments.