Virus‐like particles (VLPs) have shown great potential as biopharmaceuticals in the marketand in clinics. Nonenveloped, in vivo assembled VLPs are typically disassembled andreassembled in vitro to improve particle stability, homogeneity, and immunogenicity. At theindustrial scale, cross‐flow filtration (CFF) is the method of choice for performingreassembly by diafiltration. Here, we developed an experimental CFF setup with an on‐linemeasurement loop for the implementation of process analytical technology (PAT). Themeasurement loop included an ultraviolet and visible (UV/Vis) spectrometer as well as alight scattering photometer. These sensors allowed for monitoring protein concentration,protein tertiary structure, and protein quaternary structure. The experimental setup wastested with three Hepatitis B core Antigen (HBcAg) variants. With each variant, threereassembly processes were performed at different transmembrane pressures (TMPs).While light scattering provided information on the assembly progress, UV/Vis allowed formonitoring the protein concentration and the rate of VLP assembly based on themicroenvironment of Tyrosine‐132. VLP formation was verified by off‐line dynamic lightscattering (DLS) and transmission electron microscopy (TEM). ... mehrFurthermore, the experi-mental results provided evidence of aggregate‐related assembly inhibition and showedthat off‐line size‐exclusion chromatography does not provide a complete picture of theparticle content. Finally, a Partial‐Least Squares (PLS) model was calibrated to predict VLPconcentrations in the process solution.Q²values of 0.947–0.984 were reached for thethree HBcAg variants. In summary, the proposed experimental setup provides a powerfulplatform for developing and monitoring VLP reassembly steps by CFF.