Biomaterial-based bioinks are increasingly utilized in bioprinting to engineer three-dimensional (3D) constructs with living cells for tissue engineering and disease modeling. Among various bioinks explored, alginate-based formulations stand out due to their good biocompatibility, mild gelation conditions, tunable mechanical properties, and ease of crosslinking via divalent cations such as calcium. Despite their widespread use, standardized protocols for preparing alginate-based bioinks and characterizing bioprinted constructs have not been well documented. Our laboratory has developed and validated reproducible methods for preparing a variety of alginate-based bioinks and printing cell-laden constructs tailored for diverse applications. In this article, we present detailed step-by-step protocols covering bioink preparation and rheological characterization, extrusion-based bioprinting of cell-laden constructs, post-printing culture and co-culture techniques, printability assessment, and live/dead and immunofluorescence assays. These protocols serve as a standardized framework for the fabrication and characterization of 3D bioprinted alginate-based cell-laden constructs, thereby facilitating translational research in tissue engineering, disease modeling, and preclinical therapeutic development. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Bioink preparation Basic Protocol 2: Bioink characterization using rheology Basic Protocol 3: Scaffold design and bioprinting Support Protocol: 3D-printing parameter determination Basic Protocol 4: Printability and cell viability analyses, and immunofluorescence assay.
