<p>Spatial cues and cell–cell interactions within in vivo tissue architecture generate physical signals that regulate cell behavior. These mechanical interactions drive processes such as morphogenesis, homeostasis, regeneration and disease progression. To recapitulate these spatial niches in vitro, lithographic micropatterning allows for precise control over the geometry, composition, topography and mechanics of cell-adhesive substrates. Micropatterned cultures have been essential in uncovering key mechanotransduction mechanisms, including YAP/TAZ-mediated signaling, mechanoregulation in cancer, aging and early embryonic development. However, accessible and adaptable micropatterning protocols for conventional cell biology laboratories remain limited. In this protocol, we present a comprehensive procedure for generating flexible, high-resolution micropatterns (down to 10 × 10 µm²), optimized for high-magnification confocal imaging, long-term cell culture, customizable functionalization (proteins, peptides or both) and extended shelf life. This versatile and convenient procedure can be performed by trained PhD students or postdoctoral researchers, does not require prior expertise in photolithography and can be completed within 2 d. In addition, we provide a step-by-step guide to study mechanotransduction using YAP/TAZ functional readouts that can be completed within 5 d. This protocol offers an affordable and scalable solution applicable to a wide range of biological questions, allowing for diverse experimental needs, and represents a valuable tool for advancing mechanobiology and other areas of cell biology.</p>