Wound healing of soft tissue and bone defects is a complex process in which cellular differentiation and adaption are regulated by internal and external factors, among them are many different proteins. In contrast to insights into the significance of various single proteins based on model systems, the knowledge about the processes at the actual site of wound healing is still limited. This is caused by a general lack of methods that allow sampling of extracellular factors, metabolites, and proteins in situ. Sampling of wound fluids in combination with proteomics and metabolomics is one of the promising approaches to gain comprehensive and time resolved data on effector molecules. Here, we describe an approach to sample metabolites by microdialysis and to extract proteins simultaneously by adsorption. With this approach it is possible (i) to collect, enrich, and purify proteins for a comprehensive proteome analysis; (ii) to detect more than 600 proteins in different defects including more than 100 secreted proteins, of which many proteins have previously been demonstrated to have diagnostic or predictive power for the wound healing state; and (iii) to combine continuous sampling of cytokines and metabolites and discontinuous sampling of larger proteins to gain complementary information of the same defect.