Immunoassays are used to quantify molecules of biological interest based on the specificity and selectivity of antibody reagents generated. In HTS and lead optimization projects, assays are designed to detect molecules that are produced intracellularly or secreted in response to compounds screened. This chapter describes the basics of designing and implementing robust, automation friendly immunoassays for HTS, modes of immunoassay formats (competitive and sandwich), instrumentation, reagent selection, experimental design and detailed data analysis concepts. The importance of an appropriate curve-fitting model for calibration curves used for quantification is also addressed in detail. This is an excellent primer for beginners as well as for experienced investigators.