Isoelectric Focusing (IEF) Coupled with Mass Spectrometry (IEF-MS) is a powerful analytical approach that combines the high-resolution separation of proteins by their isoelectric points with the precise molecular identification capabilities of mass spectrometry. After proteins are focused and separated based on pI, they are directly introduced into a mass spectrometer, enabling detailed characterization of protein isoforms, post-translational modifications, and complex mixtures. This coupling enhances proteomic analyses by providing both separation and structural information in a single workflow, making it invaluable for biomarker discovery, biopharmaceutical development, and clinical research.