Fourier Transform Infrared (FTIR) has been use to discriminate different pathogens by signals from cells infected with these versus normal cells as references. To do the statistical analysis, Partial Least Square Regression (PLSR) was utilized to distinguish any two kinds of virus‐infected cells and normal cells. Validation using Bootstrap method and Cross‐validations were employed to calculate the shrinkages of Area Under the ROC Curve (AUC) and specificities corresponding to 80%, 90%, and 95% sensitivities. The result shows that our procedure can significantly discriminate these pathogens when we compare infected cells with the normal cells. On the height of this success, PLSR was applied again to simultaneously compare two kinds of virus‐infected cells and the normal cells. The shrinkage of Volume Under the Surface (VUS) was calculated to do the evaluation of model diagnostic performance. The high value of VUS demonstrates that our method can effectively differentiate virus‐infected cells and normal cells.