BACKGROUND: Although, it is well known that various types of anticoagulant influence on plasma biochemistry in clinical chemistry analysis, the evaluation of such an effect on IR spectrum of plasma has not been reported so far. OBJECTIVE: In this study, we compare FTIR spectra of plasma films from healthy mice C57Bl/6J, which were collected by application of various types of anti-clotting agents such as low molecular heparin, trisodium citrate, EDTA and aspisol. METHODS: FTIR spectra were recorded using Fourier Transform Infrared imaging and evaluated with a support of Principal Component Analysis (PCA). RESULTS: We found significant changes in spectral profile of plasma among three groups of anticoagulants: (1) nadroparine administrated intraperitoneally and blood collected by using aspisol, (2) citrate, and (3) EDTA, heparin and EDTA/aspisol. The spectral variation is mainly found in the regions associated with proteins and lipids. CONCLUSIONS: Spectral features found in this study indicate the alternation in structure of plasma proteins as well as lipids/fatty acids. This suggests a careful choice of the anticoagulant in clinical/pharmacological studies on plasma with the use of FTIR spectroscopy.