Multivariate data analysis techniques are ideal to decrypt chemical differences between anatomical features or tissue areas in hyperspectral images of biological samples. This protocol provides a user-friendly pipeline and graphical user interface (GUI) for data pre-processing and un-mixing of pixel spectra into their contributing pure components by multivariate curve resolution-alternating least squares (MCR-ALS) analysis. The analysis considers the full spectral profile to identify the chemical compounds and to visualize their distribution across the sample to categorize chemically distinct areas. Results are rapidly achieved (usually less than 30 - 60 min/image) and are easy to interpret and evaluate both in terms of chemistry and biology, making the method generally more powerful than principal component analysis (PCA) or single band intensity heap maps. In addition, chemical and biological evaluation of the results by means of reference matching and segmentation maps (based on k-means clustering) are possible.