Objective: The purpose of this study was to analyze the viability of human oral fibroblasts exposed to the action of 2% lidocaine, using DNA quantification and cell metabolic activity methods. Methods: Human oral fibroblasts were cultured in microplates of 96 wells with DMEM supplemented with FBS 10% in a concentration of 10.000 cells/100 µl of medium. The cells were incubated in a 5% of CO2 at 37º C for 24 hours. Subsequently, cultured cells were washed with PBS and exposed to different concentrations (0,05, 0,5, 1, 5, 10, 25, 50, 75 and 100%) of lidocaine 2% with epinephrine 1:100.000 diluted in DMEM free of FBS for 30 minutes. The quantification of DNA released in the culture medium was performed using a spectrophotometer. The analysis of cell metabolic activity was carried out with WST-1 assay. In each well, the reagent were added and the cells were incubated for 4 hours. The quantification of the cell metabolic activity was performed by using a microplate Reader ELX800. The controls groups were cells that did not receive treatment and cells treated with Triton X-100 1% solution. Results: The results revealed a progressive release of DNA to the culture medium and a reduction of cell metabolic activity that are directly proportional to the increase of the lidocaine concentrations. In both cases, lidocaine concentration above 50% demonstrated to be very cytotoxic with 100% of cell mortality. For the contrary, concentrations up to 5% resulted in insignificant cell mortality rates. Conclusion: There is a dose-dependent cytotoxic effect of 2% lidocaine on cultured human oral fibroblasts at 30 minutes. We would therefore recommend the use of 5% dilutions of the product in human tissues. This work was supported by FIS PI08/165 from the Spanish Fondo de Investigaciones Sanitarias