AbstractInterstitial fluid (ISF) that surrounds cells in tissues of the body is a novel source of biomarker that complements conventional sources like blood, urine, and saliva. To overcome difficulties in harvesting ISF, a minimally invasive, rapid, simple‐to‐use, cost‐effective method is developed to collect ISF from the skin involving a microneedle (MN) patch. By pressing 650 µm long MNs at an angle just below the skin surface, blood‐free ISF flows through micropores to the skin surface and is absorbed into a thin strip of paper on the MN patch backing for subsequent analysis. An optimized method in rat skin in vivo is well tolerated and able to collect >2 µL of ISF within 1 min. Brief skin pretreatment with MNs followed by a 5 min delay dramatically increases subsequent ISF collection by a mechanism believed to involve increased skin hydration. ISF collection using an MN patch has the potential to simplify access to biomarkers in ISF for research and future medical diagnostic and monitoring applications.