Autoantigen microarrays are being used increasingly to study autoimmunity. Significant variation has been observed when comparing microarray surfaces, printing methods, and probing conditions. In the present study, 24 surfaces and several arraying parameters were analyzed using >500 feature autoantigen microarrays printed with quill pins. A small subset of slides, including FAST, PATH, and SuperEpoxy2, performed well while maintaining the sensitivity and specificity of autoantigen microarrays previously demonstrated by our laboratory. By optimizing the major variables in our autoantigen microarray platform, subtle differences in serum samples can be identified that will shed light on disease pathogenesis.