This article presents an overview of the literature and a review of recent advances in the analysis of stable and transient protein–protein interactions (PPIs) with a focus on their function within cells, organs, and organisms. The significance of PTMs within the PPIs is also discussed. We focus on methods to study PPIs and methods of detecting PPIs, with particular emphasis on electrophoresis-based and MS-based investigation of PPIs, including specific examples. The validation of PPIs is emphasized and the limitations of the current methods for studying stable and transient PPIs are discussed. Perspectives regarding PPIs, with focus on bioinformatics and transient PPIs are also provided.