The enzyme-linked immunosorbent assay (ELISA) is a simple and rapid technique for detecting and quantitating antibodies or antigens attached to a solid surface. Being one of the most sensitive immunoassays, ELISA offers commercial value in laboratory research, diagnostic of disease biomarkers, and quality control in various industries. This technique utilizes an enzyme-linked antibody binding to a surface-attached antigen. Subsequently, a substrate is added to produce either a color change or light signal correlating to the amount of the antigen present in the original sample. This chapter provides the procedures required for carrying out indirect ELISA, one of the many forms of ELISA, to detect polystyrene-immobilized antigen. Methodological approaches to optimize this assay technique are also described, a prerequisite for automation and multiplexing.