ELISA systems represent highly sensitive analytical methods and have long been established and proven in many areas. Examples include clinical diagnostics, veterinary diagnostics, or food and environmental analytics. To create the conditions for optimally optimized and therefore stable ELISA systems already in development, key reagents such as antibodies or conjugates must be produced and tested for their suitability in the respective ELISA. “Auxiliary reagents” such as buffer solutions, standard solutions with defined analyte concentration, or blocking solutions are to be optimized and tested in the respective assay. This chapter is intended to provide assistance on which factors in ELISA development can be decisive, but also problematic, and which sources of error may come into question when establishing assays.