Within recent years, proteomics has become a consolidated field in terms of the amount of analysis of both clinical and basic research samples. Historically, the preferred sample format has been liquid-based, and the analysis has been based on a combination of liquid chromatography coupled to a mass spectrometer. The main limitation of the conventional large-scale proteomic approach is the loss of the spatial location of the analytes due to intrinsic sample preparation. In this respect, mass spectrometry imaging (MSI) provides a unique opportunity to access protein analysis down to 10 µm spatial resolution in a routine manner. However, it is noteworthy that despite the latest advances in the MSI field in both hardware and software as well as in sample preparation methods, deep proteomics analysis at the desired spatial lateral resolution is not possible. To bridge the gap between deep proteomic analysis and MSI, spatially-resolved proteomic strategies were developed. There are several strategies for spatially-resolved proteomic that range from the analysis of intact protein to different combinations of “on tissue” digestion and even microextractions that increase the analytical resolution power of this technique. This chapter gives an overview of the path that the proteomics field has taken in MSI to date, as well as the latest developments that are taking place and an introduction of some of the basic workflows for sample preparation.