Mycoplasma pneumoniae is a cell wall-deficient prokaryote, mainly known to colonize the human respiratory tract and to be endemic, with epidemic peaks every 6 years, in older children and young adults. Diagnosis of M. pneumoniae is challenging because of the fastidious nature of the pathogen and the possibility of asymptomatic carriage. Laboratory diagnosis of M. pneumoniae infection based on antibody titration in the serum samples of patients remains the most practiced method. Because of the potential problem of immunological cross-reactivity with the use of polyclonal serum for M. pneumoniae, an antigen-capture enzyme-linked immunosorbent assay (ELISA) has been developed to improve the specificity of serological diagnosis. ELISA plates are coated with M. pneumoniae polyclonal antibodies, raised in rabbits and rendered specific after adsorption against a panel of heterologous bacteria that share antigens with M. pneumoniae species and/or are known to colonize the respiratory tract. The reacted M. pneumoniae homologous antigens are then specifically recognized by their corresponding antibodies in the serum samples. Further optimization of the physicochemical parameters to which the antigen-capture ELISA is subjected led to a highly specific, sensitive, and reproducible ELISA.