The Moderate Resolution Imaging Spectroradiometer (MODIS) instrument on-board NASA’s Earth Observing System Aqua satellite has continued providing Earth-science measurements for over two decades. Understanding the changes in instrument response throughout this extended mission is critical to providing reliable, quality data to the science community. To assist in this understanding, MODIS is equipped with on-board calibrators including the unique spectro-radiometric calibration assembly (SRCA). The SRCA can be commanded to provide calibrated signals to measure the radiometric response, spatial alignment, and spectral behavior of MODIS detectors. Using the SRCA in its spatial configuration provides the ability to characterize the relative registration of all 36 MODIS spectral bands, in both along-scan and along-track directions. The spectral configuration leverages an internal monochromator to measure the relative spectral response functions (RSRs) of MODIS detectors from 400 nm to 1 μm. This paper provides a comprehensive review of the SRCA spatial and spectral calibrations, their traceability to pre-launch measurements, performance against specifications, and investigation of measurement uncertainties. Recently, the SRCA was commanded into a special configuration to investigate signal cross-contamination (crosstalk) among MODIS bands. Measurements and lessons learned from this testing are also included.