The deposition of affinity reagents on to spatially discrete solid substrates is utilized for constructing protein microarrays. Protein microarrays have found their way into various applications within characterisation of proteins, affinity binders, and diseases, for diagnostics and biomarker discovery purposes. The most common methods of producing protein microarrays are by immobilizing the proteins on a solid support consisting of either microspheres or functionalized planar microscope slides, both of which have different pros and cons as pertaining to throughput, storage stability, accessibility, etc. For extensive collections of proteins, the planar format is often preferred as planar substrates, such as functionalized microscope slides, can hold multiple tens of thousands of different proteins if arrayed at a high enough density. In this chapter, we detail our production of high-density planar protein microarrays comprising of 42,100 protein fragments derived from 18,955 Ensembl Gene ID (Ensembl Release 112 (May 2024)).