Direct protein expression has been developed for in-cell NMR applications in several types of eukaryotic cells, ranging from yeast to cultured human cells, in parallel with the approaches relying on exogenous protein delivery. Protein expression allows maturation processes to be monitored starting right after protein synthesis. In this chapter, the existing approaches are described, with a particular focus on protein expression in human cells for in-cell NMR, which was developed by the authors of this chapter and was applied to investigate different cellular processes.