The elucidation of the subcellular localization of proteins is very important in order to deeply understand their functions. In fact, proteins activities are strictly correlated to the cellular compartment and microenvironment in which they are present.In recent years, several effective and reliable proteomics techniques and computational methods have been developed and implemented in order to identify the proteins subcellular localization. This process is often time-consuming and expensive, but the recent technological and bioinformatics progress allowed the development of more accurate and simple workflows to determine the localization, interactions, and functions of proteins.In the following chapter, a brief introduction on the importance of knowing subcellular localization of proteins will be presented. Then, sample preparation protocols, proteomic methods, data analysis strategies, and software for the prediction of proteins localization will be presented and discussed. Finally, the more recent and advanced spatial proteomics techniques will be shown.