This guide outlines a step-by-step methodology for utilizing Nucleic Acid Programmable Protein Arrays (NAPPA) to identify and characterize novel autoantibody biomarkers for specific diseases. The guide starts with a detailed description of the discovery phase design, including target antigens, sample selection, and experimental controls. It further describes the protein microarray fabrication, emphasizing quality control measures to ensure protein expression and reliable array performance. The third section describes the autoantibody discovery and validation process, including sample preparation, data acquisition, and processing. Finally, it offers a range of data analysis strategies for identifying biomarkers using the NAPPA for further investigation.