Eukaryotic cells possess the remarkable ability to sense and respond to mechanical cues from their extracellular environment, a phenomenon known as mechanobiology, which is crucial for the proper functioning of biological systems. Micropillars have emerged as a prominent tool for quantifying cellular forces and have demonstrated versatility beyond force measurement, including the modulation of the extracellular environment and the facilitation of mechano-stimulation. In this comprehensive review, innovative strategies in micropillars’ design, fabrication, characterization, and biosensing applications are explored. The review begins with a foundational overview of micropillar-based cell mechanobiology studies to provide a complete understanding, and then it delves into novel methodologies within each domain. The latter part of the review unveils innovative micropillars’ applications beyond mechanobiology, such as their use in enhancing biosensing surfaces and as upstream fluid manipulators for biosensors. Finally, in this review, future research directions are discussed and the current limitations of these techniques are outlined. Despite the extensive exploration of micropillar applications, a significant gap remains between research advancements and the practical implementation of micropillars in point-of-care diagnostics. Bridging this gap is crucial for translating laboratory innovations into real-world medical and diagnostic tools.