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Graphical abstract Highlights • In-flow immobilization of label-free proteins can be monitored by MRI. • MRI reveals the spatial organization of immobilized proteins in flow. • Protein concentration and flow rate control the protein spatial organization. Abstract Protein immobilization is a key enabling technology for flow biocatalysis. For this purpose, many different immobilization protocols and characterization techniques have been developed in recent decades. However, examples where proteins are directly immobilized on ready-to-use reactors are scarce, likely due to the lack of analytical tools to monitor in-flow protein immobilization in a non-invasive manner. Here, we have for the first time exploited Magnetic Resonance Imaging (MRI) to characterize in-flow protein immobilization on pre-packed bed columns. This concept was demonstrated by in-flow immobilization of a green fluorescence protein. MRI analysis revealed that both the protein concentration of the flushed solution and flow rate play key roles in controlling the spatial organization of the protein across the packed-bed reactor. This analytical tool coupled to in-flow protein immobilization has been expanded to more industrially relevant enzymes, such as the lipase from Thermomyces lanuginosus , achieving a ready-to-use reactor packed with a heterogeneous biocatalyst with high activity (up to 3000 U × g−1) and high stability (75% residual activity after 1 h incubation at 60 °C). Introducing new analytical tools during the fabrication of heterogeneous biocatalysts will contribute to make the process of immobilizing proteins on solid carriers more rational than currently is. [ABSTRACT FROM AUTHOR]