In their native state, milk lipids are present in the form of dispersed droplets called Milk Fat Globules (MFG, average diameter of 4 µm in human and bovine milks) that have been recently pointed out as key elements of early nutritional programming. The native MFG is a unique biophysical element differing from other lipoproteic objects by its external trilayered membrane inherited from its secretory past. To play its major biological function, i.e. deliver energy to the mammal newborn, the MFG has to be hydrolyzed in the gastro-intestinal tract by lipases. This hydrolysis is typically a heterogeneous phase reaction, in which the lipase has to diffuse and get adsorbed onto the lipid phase before the formation of the enzyme-substrate complex, and the onset of the proper catalytic action. Very few techniques allow assessing the mechanism involved in the enzymatic hydrolysis of complex supramolecular substrates such as milk fat globules in emulsion. To get a better insight of this mechanism, 4-D imaging of gastric and pancreatic lipases enzymes using synchrotron SOLEIL UV fluorescence microscopy was applied. This methodology allowed mapping the adsorption and diffusion of lipases onto/within human or bovine MFG or biomimetic droplets devoid of membrane proteins. These data were completed with simultaneous transmission light imaging of MFG disintegration during hydrolysis. The high sensitivity of the DISCO beamline allowed detecting the auto-fluorescence of the digestive lipases, is a unique tool to unravel their mechanisms of adsorption onto complex substrate using free-label imaging and has generated useful data for infant formula optimization.