Protein microarrays provide a versatile method for the analysis of many protein biochemical activities. Existing DNA microarray analytical methods do not translate to protein microarrays due to differences between the technologies. Here we report a new approach, ProCAT, which corrects for background bias and spatial artifacts, identifies significant signals, filters nonspecific spots, and normalizes the resulting signal to protein abundance. ProCAT provides a powerful and flexible new approach for analyzing many types of protein microarrays.