Like in chemical catalysis, there is a clear trend in biocatalysis to carry out synthetic transformationsat the manufacturing scale heterogeneously catalyzed. Recycling of insoluble catalysts is simplified, andcontinuous reactor development thus promoted. Heterogeneous biocatalysis usually involves enzymesimmobilized on mesoporous solid supports that offer a large internal surface area. Unraveling enzymebehavior under the confinement of a solid surface and its effect on the catalytic reaction in heterogeneousenvironment present longstanding core problems of biocatalysis with immobilized enzymes. Progress indeepening the mechanistic understanding of heterogeneous biocatalytic conversions is often restrainedby severe limitations in methodology applicable to a direct characterization of solid-supported enzymes.Here we highlight recent evidence from the analysis of protein distribution on porous solid supportusing microscopic imaging methods with spatiotemporal resolution capability. We also show advancein the use of spectroscopic methods for the analysis of protein conformation on solid support. Methodsof direct characterization of activity and stability of immobilized enzymes as heterogeneous biocatalystsare described and their important roles in promoting rational biocatalyst design as well as optimizationand control of heterogeneously catalyzed processes are emphasized.