This paper presents an optimized procedure for assessing an immune-mediated cytotoxicity, produced after the addition of human and baboon serum to transgenic porcine fibroblasts. This procedure is performed with the xCELLigence Real-Time Cell Analyzer (RTCA). The xCELLigence system measures the impedance variations in the culture media of a 96-well microelectronic plate, and shows the changes in cell number and morphology in a real-time plot. However, different factors need to be optimized before developing an RTCA assay. Thus, we studied the influence of several variables, such as the number of cells seeded, the time the cells were allowed to grow before the tests, the serum concentration and the addition of rabbit complement. The findings were confirmed by the WST-1 classical cytotoxicity test. The results showed that 7.5 × 10³ cells seeded per well produced the adequate CI in 10 h. The area under the curve and the CI_min versus concentration values showed a very high correlation index (r² = 0.966 and r² = 0.92 for the first 50 h after challenge, respectively), proving that CI variations are directly proportional to the quantity of serum added. The addition of complement resulted in lower CI_min values. Therefore, both the cytolysis level with and without exogenous complement addition had to be assessed. There was a high correlation between the relative cytotoxicity assessed by WST-1 and the CI obtained by RTCA when exogenous complement was not added (r² = 0.827; p < 0.001). The correlation was average when rabbit complement was added (r² = 0.523; p = 0.046). In conclusion, culture conditions have an important influence on RTCA cytotoxicity assays.