Protein biochips have a great potential in future parallel processing of complex samples, as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high-throughput protein expression, large-scale analysis of proteins and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of Alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigene(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we have profiled the autoantibody repertoire of sera from Alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared to control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset were generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel Alopecia areata associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis, or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using Alopecia areata as a model for a autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens, as well as in diagnosis such as to differentiate Alopecia areata from other skin diseases.