This chapter describes methods for the use of antibody microarrays with rolling-circle amplification (RCA). The methods are divided into three sections. The first section covers antibody preparation and microarray production, the second describes the method for using biological samples on antibody microarrays, and the third describes the method for RCA use on antibody microarrays. RCA can be used on antibody microarrays to increase the signal from each antibody spot and lower the detection limits of the assays. We also describe a practical method for running multiple, low-volume microarrays on a single microscope slide. These methods should be useful for researchers interested in rapidly developing and optimizing custom immunoassays for the analysis of low-abundance analytes using low sample volumes.