SPOP (Speckle-type POZ protein) is an E3 ubiquitin ligase adaptor protein that mediates the ubiquitination of several substrates. Furthermore, SPOP is responsible for the regulation of both degradable and nondegradable polyubiquitination of a number of substrates with diverse biological functions. The recognition of SPOP and its physiological partners is mediated by two protein–protein interaction domains. Among them, the MATH domain recognizes different substrates, and it is critical for orchestrating diverse cellular pathways, being mutated in several human diseases. Despite its importance, the mechanism by which the MATH domain recognizes its physiological partners has escaped a detailed experimental characterization. In this work, we present a characterization of the binding mechanism of the MATH domain of SPOP with three peptides mimicking the phosphatase Puc, the chromatin component MacroH2A, and the dual-specificity phosphatase PTEN. Furthermore, by taking advantage of site-directed mutagenesis, we address the role of some key residues of MATH in the binding process. Our findings are briefly discussed in the context of previously existing data on the MATH domain.