This study investigated the in vitro effect of various vital dyes in common clinical use on human Müller cell viability, and it compared the toxicity of these dyes using a cell culture model. Müller cells were exposed to a series of concentrations (1 %, 0.5 %, 0.25 %, and 0.125 % or 12.9 mM, 6.45 mM, 3.22 mM and 1.61 mM) of Indocyanine green (ICG) for 2, 24, 48, and 72 h. Similarly, groups of Müller cells were stained with “Heavy” brilliant blue G (HBBG), Trypan blue (TB) (0.15 %, or 1.56 mM), Membrane-blue-dual (MBD), and ICG (0.25 %, or 3.22 mM) or BBG (0.025 %, or 0.3 mM) with glucose (GS) (50 %, 66 % and 75 % or 2.78 M, 3.67 M and 4.17 M) for 30, 60, and 120 s. Cell viability was measured with the Cell Counting Kit-8 (CCK-8) and Lactate Dehydrogenase (LDH) release assays. We found that high stain concentration and long exposure time resulted in increased toxicity to Müller cells. Nevertheless, ICG seemed to be safe at the clinically relevant concentration of 0.25 % (3.22 mM) in the short time of exposure. TB was safer than both HBBG and MBD, especially HBBG. Hypertonic GS as a dilution was not safe for Müller cells, and the negative effect was more obvious in 0.025 % (0.3 mM) BBG than that in 0.25 % (3.22 mM) ICG. This is the first report to observe the cytotoxicity of commonly used stains in clinical on human Müller cells in vitro, and to provide some basis for further studies, including in vivo investigation.