The Bacterial reverse mutation (AMES) assay is crucial for detecting the mutagenic potential of chemicals using bacterial strains. The solubility of the test substance is key to achieving the recommended concentration for the assay. DMSO and water are typically preferred solvents due to their compatibility and historical data. The chosen solvent must not react with the test substance and must support bacterial survival and S9 activity. Selecting a solvent compatible with Salmonella typhimurium and Escherichia coli WP2 uvrA strains, considering a maximum cytotoxic concentration of 5 mg/plate, is challenging for genetic toxicologists. This study evaluated various solvents, including N,N-Dimethyl formamide, Acetone, Acetonitrile, Ethyl acetate, 95% Ethanol, Dimethylene Glycol Monomethyl ether, Methanol, P-Dioxane, Tetrahydrofuran, and Dimethyl acetamide. Results showed that all solvents, except Tetrahydrofuran, were compatible up to 100 µL/plate or more, as they did not inhibit bacterial growth or alter bacterial revertant colony counts, making them suitable for use in the Ames assay.