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Summary: Quantification of nuclear stiffness is challenging for cells encapsulated within a 3D extracellular matrix (ECM). Here, we describe an experimental setup for measuring microenvironment-dependent tuning of nuclear stiffness using an atomic force microscope (AFM). In our setup, ECM-coated polyacrylamide hydrogels mimic the stiffness of the microenvironment, enabling the measurement of nuclear stiffness using an AFM probe in live cancer cells.For complete details on the use and execution of this protocol, please refer to Das et al. (2019) (https://doi.org/10.1016/j.matbio.2019.01.001).