The inability to reliably measure cell viability in engineered tissues remains a major roadblock to the regenerative medicine and tissue engineering industries. Cell viability is a commonly measured product attribute for therapeutic devices that contain cells. Cell viability assays were designed for use in flat culture systems, where the diffusion of assay components is not impeded by the scaffold structure. To assess the ability of an ATP assay system to reliably measure cellular ATP during cell culture in a VitroGel hydrogel scaffold, VitroGel was disassembled by shear thinning, which enables cells to be released and assayed in the absence of scaffold in order to confirm results conducted on cells in scaffolds. Various aspects of the hydrogel system and ATP assay system were varied to assess their impact on assay response. The composition of the medium used for the standard curve has a large influence on assay results. When run on gels spiked with ATP, the results were lower than controls without VitroGel. However, a 30 min pre-incubation step allows the diffusion of the assay components to reach an equilibrium so that the results were similar to the controls without gels. When gels spiked with ATP were disassembled by shear thinning, the ATP recovery was 100% as compared to controls without gel. The VitroGel and ATP assay are a reliable system for assessing measurements of cell viability in scaffolds.