Background: During negative pressure wound therapy (NPWT), open wounds are draped with a nontransparent sponge, making daily wound evaluation impossible. Sometimes, late or undetected bacterial infections and postoperative bleeding result in repetitive surgery, thus prolonging inpatient time. With the introduction of additional fluid instillation (NPWTi), the wound surface is rinsed, and bacteria, proteins and biomarkers are flushed into a collecting canister, which is later discarded. Methods: The aim of this pilot study was to analyze rinsing fluid samples (0.9% sodium chloride) from the NPWTi device in patients with acute and chronic wounds. In 31 consecutive patients a standardized laboratory analysis was performed to evaluate cellular composition and potassium, phosphate, lactate dehydrooxygenase, pH and total protein levels. Results: While there was an increase in the total cellular amount and the number of polymorphonuclear cells, the number of red blood cells (RBC) decreased after surgery. Potassium and pH showed no significant changes in the first three postoperative days, whereas total protein showed an undulant and partially significant course. Conclusion: We were able to quantify cellular metabolites by analyzing the rinsing fluid of NPWTi. We propose the analysis of this material as a novel and potentially promising tool to monitor wound status without removal of the dressing. The establishment of reference values might help to improve the NPWTi therapy.