SUMOylation contributes to the regulation of many essential cellular factors. Diverse techniques have been used to explore the functional consequences of protein SUMOylation. Most approaches consider the identification of sequences on substrates, adaptors, or receptors regulating the SUMO conjugation, recognition, or deconjugation. The large majority of the studied SUMOylated proteins contain the sequence [IVL]KxE. SUMOylated proteins are recognized by at least 3 types of hydrophobic SUMO-interacting motifs (SIMs) that contribute to coordinate SUMO-dependent functions. Typically, SIMs are constituted by a hydrophobic core flanked by one or two clusters of negatively charged amino acid residues. Multiple SIMs can integrate SUMO binding domains (SBDs), optimizing binding, and control over SUMO-dependent processes. Here, we present a survey of the methodologies used to study SUMO-regulated functions and provide guidelines for the identification of cis and trans sequences controlling SUMOylation. Furthermore, an integrative analysis of known and putative SUMO substrates illustrates an updated landscape of several SUMO-regulated events. The strategies and analysis presented here should contribute to the understanding of SUMO-controlled functions and provide rational approach to identify biomarkers or choose possible targets for intervention in processes where SUMOylation plays a critical role.