This article discusses the application of Fourier Transform Infrared (FTIR) spectroscopy in the analysis of biological materials, particularly focusing on its utility in cytological and histological diagnosis through spectral imaging. The authors highlight the importance of spectral pre-processing and data analysis, which are crucial for obtaining objective and reliable results. They describe a standardized protocol for collecting IR spectra and images from various biological samples, including fixed cytology and tissue sections, live cells, and biofluids. The protocol covers instrumental options, sample preparation, spectral acquisition, and data processing, including quality control, spectral pre-processing, feature extraction, and classification. The article also emphasizes the importance of optimizing spectral quality, minimizing water vapor interference, and selecting appropriate detectors and software for effective analysis. Additionally, it provides guidelines for sample preparation, spectral acquisition, and data processing, along with specific examples and troubleshooting tips. The authors aim to provide a comprehensive resource for researchers to conduct high-quality IR spectroscopy experiments in the biomedical field and beyond.This article discusses the application of Fourier Transform Infrared (FTIR) spectroscopy in the analysis of biological materials, particularly focusing on its utility in cytological and histological diagnosis through spectral imaging. The authors highlight the importance of spectral pre-processing and data analysis, which are crucial for obtaining objective and reliable results. They describe a standardized protocol for collecting IR spectra and images from various biological samples, including fixed cytology and tissue sections, live cells, and biofluids. The protocol covers instrumental options, sample preparation, spectral acquisition, and data processing, including quality control, spectral pre-processing, feature extraction, and classification. The article also emphasizes the importance of optimizing spectral quality, minimizing water vapor interference, and selecting appropriate detectors and software for effective analysis. Additionally, it provides guidelines for sample preparation, spectral acquisition, and data processing, along with specific examples and troubleshooting tips. The authors aim to provide a comprehensive resource for researchers to conduct high-quality IR spectroscopy experiments in the biomedical field and beyond.