The objective of this study was to examine the validity of estimating cell number using the water-soluble tetrazolium salts-8 (WST-8) assay for normal cells. The changes in mitochondrial activity of human umbilical vein endothelial cells (HUVECs) cultured under various culture conditions were evaluated using this assay. We found that cell counting of HUVECs by the WST-8 assay was not accurate under certain culture conditions, because the mitochondrial activity per cell changed depending on culture duration and medium composition. When charcoal/dextran-treated fetal bovine serum (FBS) -supplemented medium was used in cultures, the mitochondrial activity per cell was similar both in the presence and in the absence of vascular endothelial growth factor (VEGF). Furthermore, the mitochondrial activity did not affect the proliferative activity of cells, because we observed VEGF-dependent proliferation of HUVECs in this medium. For precise evaluation of cell number using the WST-8 assay, it is important to consider the accuracy of the assay results. Thus, a reliable method of cell counting using WST-8 needs to be developed.