At the foundation of many cellular processes as well as a large number of diseases is the (mis)folding of important intrinsically disordered proteins (IDPs). Despite tremendous scientific efforts, the factors driving their structural changes within the cellular context remain poorly understood. In-cell NMR spectroscopy enables investigation of IDPs directly in the living eukaryotic cell enabling investigation of its intermolecular interactions and ensuing modifications at an unprecedented atomic resolution. In the following protocol, we describe how to prepare in-cell NMR samples of IDPs within eukaryotic cells and how to measure these in-cell NMR samples of an IDP in its natural environment, the living mammalian cell. Furthermore, we outline a procedure to assess the intracellular recombinant protein concentration of the studied IDP based on in-cell NMR methods. We use α-synuclein as a model protein, but the presented approach is highly modular and therefore should be easily adapted and altered to the desired needs for the studies of different IDPs.