Microarray technology has made it possible to simultaneously study the abundance, interactions, and functions of potentially tens of thousands of biological molecules. From its earliest use in DNA microarrays, where only nucleic acids were captured and detected on the arrays, applications of microarrays now extend to those involving biomolecules such as antibodies, proteins, peptides, and carbohydrates. In contrast to the relative robustness of DNA microarrays, the use of such chemically diverse biomolecules on microarray formats presents many challenges in their fabrication as well as application. Among the many methods that have been proposed to overcome these challenges, DNA-directed assembly (DDA) has emerged as a promising strategy for the high sensitivity and multiplexed capture and detection of various analytes. In this review, we explore the challenges faced during the design, fabrication, and utilization of protein microarrays and highlight how DDA strategies, together with other recent advances in the field, are accelerating the development of platforms available for protein microarray applications.