ELISA quantitation, like any other highly sensitive bioanalytical quantitation, should be handled with care and precision. To begin with, we must review proper calibration and verification of instrumentation, pipettes, and other equipment before use. Next, we must intensively train all ELISA lab personnel in protocol reproducibility, pipetting techniques, and pipette tip handling to perform ELISA Method Development and Validation. Generally, ELISA analysis plate readouts and incubation steps are highly time-dependent. Thus, we must emphasize that plates must be analyzed quickly following the addition of substrate for any pre-reads or final reads after stopping the reaction. We can automate sample analysis for ELISA assay with liquid handlers, plate washers, and other robotics adaptable to 96- or 384-well plates.