Abstract Digital light processing (DLP) bioprinting, which provides predominant speed, resolution, and adaptability for fabricating complex cell-laden three-dimensional (3D) structures, requires a combination of photoinitiator (PI) and UV absorber (UA) that plays critical roles during the photo-polymerization of bioinks. However, the PI and UA combination has not been highlighted for cell-based DLP bioprinting. In this study, the most used PIs and UAs in cell-based bioprinting were compared to optimize a combination that can ensure the maximum DLP printability, while maintaining the cellular activities during the process. The crosslinking time and printability of PIs were assessed, which are critical in minimizing the cell damage by the UV exposure during the fabrication process. On the other hand, the UAs were evaluated based on their ability to prevent the over-curing of layers beyond the focal layer and the scattering of light, which are required for the desirable crosslinking of a hydrogel and high resolution (25–50 µms) to create a complex 3D cell-laden construct. Lastly, the cytotoxicity of PIs and UAs was assessed by measuring the cellular activity of 2D cultured and 3D bioprinted cells. The optimized PI and UA combination provided high initial cell viability (>90%) for up to 14 days in culture and could fabricate complex 3D structures like a perfusable heart-shaped construct with open vesicles and atriums. This combination can provide a potential starting condition when preparing the bioink for the cell-based DLP bioprinting in tissue engineering applications.