The EO-1 Advanced Land Imager (ALI) is the first earth-orbiting instrument to be flown under NASA's New Millenium program. The ALI employs novel wide-angle optics and a multispectral and panchromatic spectrometer. EO-1 is a technology verification project designed to demonstrate comparable or improved Landsat spatial and spectral resolution with substantial mass, volume, and cost savings. This paper provides an overview of in-flight calibration and performance assessment of the Advanced Land Imager. Included are techniques for calibrating and assessing focus and MTF using long, straight, man-made objects and monitoring of radiometric linearity and offsets using an internal calibration source, standard Earth reference scenes, and solar and lunar observations.