The analysis of the proteome and the interactome would be useful for a better understanding of the pathophysiology of several disorders, allowing the identification of potential specific markers for early diagnosis and prognosis, as well as potential targets of intervention. Among different proteomic approaches, high-density protein microarrays have become an interesting tool for the screening of protein-protein interactions and the interactome definition of disease-associated dysregulated proteins. This information might contribute to the identification of altered signaling pathways and protein functions involved in the pathogenesis of a disease. Remarkably, protein microarrays have been already satisfactorily employed for the study of protein-protein interactions in cancer, allergy, or neurodegenerative diseases. Here, we describe the utilization of recombinant protein microarrays for the identification of protein-protein interactions to help in the definition of disease-specific dysregulated interactomes.