The nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for several steps of the viral life cycle, and is abundantly expressed during infection, making it an ideal diagnostic target protein. This protein has a strong tendency to dimerization and interaction with nucleic acids. A native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) method was established for in-process control, in particular, to monitor product fragmentation and multimerization throughout the purification process. High titers of the nucleocapsid protein were expressed in E. coli with a CASPON tag, using a growth-decoupled protein expression system. Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of chromatographic steps. 730 mg purified NP per liter of fermentation could be produced by the optimized process, corresponding to a yield of 77%. The HIC-MALS method was used to demonstrate that the NP product can be produced with a purity of 95%. The molecular mass of the main NP fraction is consistent with dimerized protein as was verified by a complementary native size-exclusion separation (SEC)-MALS analysis. Peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay confirmed the high product purity, and the presence of a minor endogenous chaperone explained the residual impurities. The HIC-MALS method enables to monitor the purity of the product and simultaneously access its molecular mass.