•The invention of ELISA and its types are presented in a sequence.•Laboratory experiences with peptide analyses are provided.•The need to use protease inhibitors to protect peptides in peptide analyses is emphasized.•It is underlined that ELISA kit manufacturers have to standardize kits.•The significance of ELISA in peptide analyses is noted. Playing a critical role in the metabolic homeostasis of living systems, the circulating concentrations of peptides/proteins are influenced by a variety of patho-physiological events. These peptide/protein concentrations in biological fluids are measured using various methods, the most common of which is enzymatic immunoassay EIA/ELISA and which guide the clinicians in diagnosing and monitoring diseases that inflict biological systems. All the techniques where enzymes are employed to show antigen–antibody reactions are generally referred to as enzymatic immunoassay EIA/ELISA method. Since the basic principles of EIA and ELISA are the same. The main objective of this review is to present an overview of the historical journey that had led to the invention of EIA/ELISA, an indispensible method for medical and research laboratories, types of ELISA developed after its invention direct (the first ELISA method invented), indirect, sandwich and competitive methods, problems encountered during peptide/protein analyses (pre-analytical, analytical and post-analytical), rules to be followed to prevent these problems, and our laboratory experience of more than 15 years.