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Abstract Direct laser writing via two‐photon polymerization (2PP) is an emerging micro‐ and nanofabrication technique to prepare predetermined and architecturally precise hydrogel scaffolds with high resolution and spatial complexity. As such, these scaffolds are increasingly being evaluated for cell and tissue engineering applications. This article first discusses the basic principles and photoresists employed in 2PP fabrication of hydrogels, followed by an in‐depth introduction of various mechanical and biological characterization techniques used to assess the fabricated structures. The design requirements for cell and tissue related applications are then described to guide the engineering, physicochemical, and biological efforts. Three case studies in bone, cancer, and cardiac tissues are presented that illustrate the need for structured materials in the next generation of clinical applications. This paper concludes by summarizing the progress to date, identifying additional opportunities for 2PP hydrogel scaffolds, and discussing future directions for 2PP research.