Sufficient and controllable oxygen supply is essential for in vitro 3D cell and tissue culture at high cell densities, which calls for volumetric in situ oxygen analysis methods to quantitatively assess the oxygen distribution. This paper presents a general approach for accurate and precise non-contact 3D mapping of oxygen tension in high cell-density cultures via embedded commercially available oxygen microsensor beads read out by confocal phosphorescence lifetime microscopy (PLIM). Optimal acquisition conditions and data analysis procedures are established and implemented in a publicly available software package. The versatility of the established method is first demonstrated in model-assisted fluidic design of microperfused 3D printed hydrogel culture chips with the aim of full culture oxygenation, and subsequently for monitoring and maintenance of physiologically relevant spatial and temporal oxygen gradients in the 3D printed chips controlled by static or dynamic flow conditions during 3D culture.