Peptide microarrays have become increasingly accessible in recent years and as a result, more widely applied. Beyond its initial utility in substrate profiling, researchers are adopting peptide microarrays for the comparative screening of many different classes of enzymes, proteins/ proteomes and even living cells. Understanding the basis of peptide interactions at these diverse levels provides an unprecedented window into dissecting the complex cellular circuitries and molecular architectures of living systems. The peptides on the arrays may serve to sense protein activity (like substrates) or act as small molecule ligands (for potential therapeutic leads) in profiling, detection or diagnostic applications. This review will chart the progress made in peptide microarrays, with a focus on the recent advances that could impact how the field will be shaped in the coming years. These developments, along with the diminishing costs of library synthesis and growing commercial support, recognize that peptide microarrays will no longer remain just a vital research tool, but also a platform that could now be harnessed for wider drug discovery and point-of-care applications.