Modern methods have evolved in tissue engineering to evaluate cell viability (CV) in 3D scaffolds and tissues. These involve either the usage of 3D confocal laser microscopy of live or fixed tissues, or separation of cells from the tissue, either live or fixed, and then their analysis by flow cytometry. Generally, working with live cells has the disadvantage that all the scanning needs to be completed immediately at the end of an experiment. Two different approaches can be distinguished: staining intact cell membranes and staining fixed cells. The entire cytoplasm is stained with amine-reactive dyes (ARDs), these use the principle of dead cell exclusion. Here, we list and compare live-cell versus fixed-cells fluorescence-based methods and also show their limitations, especially when working with autofluorescent or cross-linking materials like silk or genipin-reinforced hydrogels. Microscopic techniques have the advantage over flow cytometry-based methods in that these provide the spatial distribution and morphology of the cells. Calcein AM combined with ethidium-homodimer works for most 3D constructs, where no strong fluorescent background is found on the tissue or scaffold. Frequently, however, concentrations and incubation times need to be adjusted for a specific tissue to ensure diffusion of dyes and optimise emittance for detection.