This article describes a protocol for using Fourier transform infrared (FTIR) spectroscopy to analyze biological materials. FTIR spectroscopy is a non-invasive, label-free technique that allows the extraction of biochemical information and imaging of tissue or cell architecture. The protocol outlines the steps for collecting IR spectra and images from biological samples, including fixed cytology and tissue sections, live cells, or biofluids. It covers instrumental options, appropriate sample preparation, different sampling modes, and important advances in spectral data acquisition. After acquisition, data processing involves quality control, spectral pre-processing, feature extraction, and classification of the data. The protocol also discusses the application of FTIR spectroscopy in cancer classification and imaging, as well as in other research areas such as environmental toxicology, consumer safety, taxonomy, and the food industry. The article provides detailed information on experimental design, including sample preparation, spectral acquisition, and data processing. It also includes information on materials, reagents, and equipment required for the protocol. The protocol is designed to standardize methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis.This article describes a protocol for using Fourier transform infrared (FTIR) spectroscopy to analyze biological materials. FTIR spectroscopy is a non-invasive, label-free technique that allows the extraction of biochemical information and imaging of tissue or cell architecture. The protocol outlines the steps for collecting IR spectra and images from biological samples, including fixed cytology and tissue sections, live cells, or biofluids. It covers instrumental options, appropriate sample preparation, different sampling modes, and important advances in spectral data acquisition. After acquisition, data processing involves quality control, spectral pre-processing, feature extraction, and classification of the data. The protocol also discusses the application of FTIR spectroscopy in cancer classification and imaging, as well as in other research areas such as environmental toxicology, consumer safety, taxonomy, and the food industry. The article provides detailed information on experimental design, including sample preparation, spectral acquisition, and data processing. It also includes information on materials, reagents, and equipment required for the protocol. The protocol is designed to standardize methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis.