Matrix assisted laser desorption/ ionization (MALDI) mass spectrometry imaging is a powerful tool that opens new research opportunities in the field of biology. In thiswork, predictive modelwas developed to discriminatemetabolic myofiber types using the MALDI spectral data. Rat skeletal muscles are constituted of type I and type IIA fiber, which have an oxidative metabolism for glycogen degradation, and type IIX and type IIB fiber which have a glycolytic metabolism, present in different proportions according to the muscle function and physiological state. So far, myofiber type is determined by histological methods that are time consuming. Thanks to the predictive model, we were able to predict not only the metabolic fiber type but also their location, on the same muscle section that was used for MALDI imaging.