HT-29 cellular proliferation was assessed at different cell densities (1250, 2500, 5000, 10000 and 20000 cells/well) with a modified Alamar Blue assay (A). The increases of fluorescence at densities of 1250–10000 cells/well indicated the fluorescence readings designate the true cell number at these cell densities. The fluorescence at 20000 cells/well tended to decrease compared to that at 10000 cells/well, but the decrease was not statistically significant (P > 0.05, NS). To validate the modified version of Alamar Blue assay and to find whether the fluorescence drop at 20000 cells/well was a feature of the assay or cellular proliferation itself, two parallel procedures were performed simultaneously. One procedure (B) was the modified procedure similar to that in the modified Alamar Blue assay. Another procedure (C) was no modified procedure. The same range of cell densities (1250, 2500, 5000, 10000 and 20000 cells/well) were used in both procedures. At the end of two procedures, the cell numbers were assessed using CellTiter-Glo luminescent cell viability assay. Luminescence increased along with the increases of cell densities at 1250–10000 cells/well for both modified (B) and no modified (C) procedures. Luminescence at 20000 cell/well tended to decrease in both procedures, although the decreases were not statistically significant compared to that at 10000 cell/well density (P > 0.05; NS). Significance value: * P <0.05; ** P < 0.001; NS P > 0.05.