Objective Evaluation of the diagnostic potential of freeze dried sera in comparison to thin dry film analysis for recording ATR-FTIR spectra. Results For this purpose, we compared our novel sample preparation technique i.e. freeze dried with conventional technique i.e. thin dry film sera. Using both methods ATR-FTIR spectra were recorded from Salmonella Typhi infected and healthy control human sera samples. When PCA was applied PC1 scores showed more inter-class variation among infected and healthy controls when freeze dried sample was used (90 %) as compared to thin dry film method (46 %). Conclusions Potential of ATR-FTIR for discrimination of bio-molecules between two classes of samples is enhanced when freeze dried sera instead of thin dry film method is used.