Atomic force microscopy (AFM) is capable of nanoscale imaging but has so far only been used on cell surfaces when applied to a living cell. Here, we describe a step-by-step protocol for nanoendoscopy-AFM, which enables the imaging of nanoscale structures inside living cells. The protocol consists of cell staining, fabrication of the nanoneedle probes, observation inside living cells using 2D and 3D nanoendoscopy-AFM, and visualization of the 3D data.For complete details on the use and execution of this protocol, please refer to Penedo et al. (2021)1 and Penedo et al. (2021).2