ISO 10993-5 provides one of the accepted standards for testing the biotoxicity of new materials. All of the recommended test procedures rely upon the uptake or metabolism of dye by living cells. Results of direct contact tests can be potentially compromised by interaction or adsorption of the dye or its metabolic products. Therefore, the aim of the current study was to validate the use of the eGFP signal of transfected NIH-3T3 fibroblasts with the results of the MTT test in order to provide a test procedure that is very close to the ISO 10993-5 but has the advantage of not relying on the addition of dye. Our tests show that the MTT assay detects cytotoxicity in the eGFP NIH-3T3 cells at least as well as in the L929 cells. To facilitate the validation, we chose to integrate the fluorescence measurements into the MTT test procedure. To that end, an additional washing step was introduced. Additionally, medium without phenol red was used, resulting in a very high correlation of both measurements. Without these modifications, the fluorescence test was comparable to the MTT test in its ability to detect the cytotoxic potential of substances; however, it did result in slightly elevated IC50 concentrations. As the results of both tests correlated highly, measurement of the eGFP signal appears to present a reliable tool for detecting cytotoxicity of materials in line with the ISO 10993-5 norm with the advantage of avoiding the addition of dyes and the subsequent potential interaction with test materials.