The enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods for the quantitative determination of proteins, hormones, and biomarkers in modern biomedicine. This article reviews the historical development of the method—from the classical work of Engvall and Perlmann (1971) to contemporary high-sensitivity digital and multiplex formats. The principles of ELISA, its main types (direct, indirect, sandwich, and competitive), as well as practical factors that influence analytical accuracy— such as matrix effects, blocking, washing, and calibration—are described. Special attention is given to recent technological advancements, including digital (Simoa) ELISA, microfluidic platforms, and multiplex panels that enable simultaneous analysis of multiple biomarkers at ultra-low concentrations. Examples are provided of ELISA applications in studies of hormonal status, inflammation, stress-induced changes, PTSD, and neurodegenerative diseases, particularly Alzheimer’s disease. The article highlights that ELISA remains the “gold standard” of biochemical diagnostics, and that ongoing developments in miniaturization, automation, and digital analysis are establishing this method as a foundation for future personalized medicine.