Pericellular pO2 (ppcO2) was compared with the release of cytoplasmatic enzyme lactate dehydrogenase (LDH) and mitochondrial metabolic function (tetrazolium salt reduction, MTT) as an alternative method to evaluate cytotoxicity. L-929 cells were seeded and incubated with 3 different control materials to test their cytotoxicity effects by these methods. High density polyethylene and copper were respectively used as negative and positive controls, while a 0.9% NaCl solution was as reagent control and extraction vehicle. PpcO2 was measured by a rod polarographic Clark-type probe connected to Licox pO2 computer (GMS mbH, Kiel, Germany). One-way ANOVA test showed significant differences among groups regarding every methods (p < 0.0005). The comparison of the mean variation coefficients of three methods showed no significant differences. In addition, a significant correlation was found between ppcO2 and MTT (r = 0.621; p < 0.001) as well as ppcO2 and LDH data (r = 0.474; p < 0.001). In conclusion, ppcO2 may be considered a reliable, safely and alternative method to test cytotoxicity.