A self-calibrating scanning system and method are used in the analysis of biomolecules on a microarray. The self-calibrating scanning system comprises an excitation light source, an optical portion, a detection portion and a calibration portion that includes a calibration apparatus and compensation portion. The calibration apparatus comprises a light source having a highly reproducible or calibrated light based on a preselected or reference light level. The calibration apparatus emits the calibrated light that is measured by the detection portion of the scanning equipment. If the detection components are stable, the components will measure a constant output value for the calibrated light over time. As a detection component changes with time, the output value will change for the same calibrated light. The method comprises the steps of initially calibrating the detection portion of the scanning system and subsequently calibrating the detection portion to compensate for sensitivity changes.