This study describes a non-invasive method for mapping interstitial fluid pressure within hydrogel-based microscale tissues. The method is based on embedding (or forming) a tissue within a silicone (PDMS) microfluidic device, and measuring the extremely slight displacement (<1 μm) of the PDMS optically when the device is pressurized under static and flow conditions. The displacement field under uniform pressure provides a map of the local device stiffness, which can then be used to obtain the non-uniform pressure field under flow conditions. We have validated this method numerically and applied it towards determining the hydraulic properties of tumor cell aggregates, blind-ended epithelial tubes, and perfused endothelial tubes that were all cultured within micropatterned collagen gels. The method provides an accessible tool for generating high-resolution maps of interstitial fluid pressure for studies in mechanobiology.