Fourier transform infrared (FTIR) microspectroscopy is a powerful tool for the study of complex biological systems. Indeed, it is employed to characterize intact cells, tissues, and whole model organisms such as nematodes, since it allows to obtain a chemical fingerprint of the sample under investigation, giving information on the molecular composition and structures. The successful application of this technique for the in situ study of biological processes requires specific sample preparations, in order to obtain reliable and reproducible results. In the present work, we illustrate the optimized procedures to prepare biological samples for IR measurements and the method to collect and analyze their FTIR spectra. In particular, we describe here the investigations on bacterial cells, intact eukaryotic cells, and whole intact nematode specimens.