The complex cellular microenvironment plays an important role in determining cell fate. For example, stem cells located in a microenvironment termed niche integrate a wide variety of extrinsic cues to take distinct fate choices. Capturing this multiple-input/multiple-output system in vitro has proven to be very challenging. In order to address this issue, we developed and validated a microfabricated cellular array platform, termed artificial niche microarrays, which is capable of performing high-throughput single-cell assays under physiologically relevant conditions. The platform allows exposing cultured cells to differential signaling cues displayed on soft hydrogel substrates having variable stiffness. The behavior of the seeded cells can be readily quantified across over 2000 multivariate microenvironments. Here we describe a pipeline for performing multifactorial, image-based assays with these artificial niche microarrays. The procedure details the steps from microarray production, cell culture, cell phenotyping, data extraction to statistical analysis.