Tolerance to organic solvents is an important characteristic for selecting bacteria that are useful for producing commodity chemicals. An assay method for tolerance should be easy to perform and differences in tolerance should be reproducible. We propose a cell growth assay using tetrazolium violet and image analysis for assessing tolerance. When five microorganisms including Escherichia coli were tested, the organic solvent tolerance (OST) of microorganisms used was rapidly and qualitatively assessed and the degrees of OST coincided with those from the conventional method using solvent overlaid-solid medium. Moreover, the proposed method was applicable to the estimation of OST for cyclohexane resistant E. coli OST3410 using various organic solvent mixtures.