The use of vibrational spectroscopy techniques, such as Fourier-transform infrared (FTIR) and Raman spectroscopy, has been a successful method to study the interaction of light with biological materials and facilitate novel cell biology analysis. Disease screening and diagnosis, microbiological studies, forensic and environmental investigations make use of spectrochemical analysis very attractive due to its low cost, minimal sample preparation, non-destructive nature and substantially accurate results. However, there is now an urgent need for multivariate classification protocols allowing one to analyse biological-derived spectrochemical data in order to obtain accurate and reliable results. This is stimulated by the fact that applications of deep-learning algorithms of complex datasets are being increasingly recognized as critical towards extracting important information and visualizing it in a readily interpretable form. Hereby, we have constructed a protocol for multivariate classification analysis of vibrational spectroscopy data [FTIR, Raman and near-infrared (NIR)] highlighting a series of critical steps, such as pre-processing, data selection, feature extraction, classification and model validation. This is an essential aspect towards the construction of a practical spectrochemical analysis model for biological analysis in real-world applications, where fast, accurate and reliable classification models are fundamental.