Epithelial cells reside on specialized extracellular matrices that provide instructive cues to regulate and support cell function. The authors have previously demonstrated that substrate topography with dimensions similar to the native extracellular matrix (submicrometer and nanoscale features) significantly impacts corneal epithelial proliferation and migration. In this work, synthetic hydrogels were modified with both topographic and biochemical cues, where specified peptide ligands were immobilized within nanopatterned hydrogels. The efficient, systematic study of multiple instructive cues (peptide, peptide concentration, topographic dimensions), however, is contingent on the development of higher throughput platforms. Toward this goal, the authors developed a hydrogel array platform to systematically and rapidly evaluate combinations of two different peptide motifs and a range of nanoscale topographic dimensions. Specifically, distinct functional pegylated peptide ligands, RGD (GGGRGDSP) and AG73 (GRKRLQVQLSIRT), were synthesized for incorporation into an inert hydrogel network. Elastomeric stencils with arrays of millimeter-scale regions were used to spatially confine hydrogel precursor solutions on elastomeric stamps with nanoscale patterns generated by soft lithography. The resulting topographically and peptide-functionalized hydrogel arrays were used to characterize single cell migration. Epithelial cell migration speed and persistence were governed by both the biochemical and topographical cues of the underlying substrate.