A novel protein microarray technology, called high-density nucleic acid programmable protein array (HD-NAPPA), enables the serological screening of thousands of proteins at one time. HD-NAPPA extends the capabilities of NAPPA, which produces protein microarrays on a conventional glass microscope slide. By comparison, HD-NAPPA displays proteins in over 10,000 nanowells etched in a silicon slide. Proteins on HD-NAPPA are expressed in the individual isolated nanowells, via in vitro transcription and translation (IVTT), without any diffusion during incubation. Here we describe the method for antibody biomarker identification using HD-NAPPA, including four main steps: (1) HD-NAPPA array protein expression, (2) primary antibodies (serum/plasma) probing, (3) secondary antibody visualization, and (4) image scanning and data processing.