Time-concentration curves for the topical anti-viral drug acyclovir can provide valuable information for drug development. Open flow microperfusion (OFM) is used for continuous sampling of dermal interstitial fluid but it requires validated methods for subsequent sample analysis. Therefore, we developed a sensitive, selective and high throughput ultra-high-performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HR MS/MS) method to determine acyclovir in human dermal interstitial fluid and serum. We validated the method over a concentration range of 0.1 - 25 ng/mL for a sample volume of just 20 μL and employed cation-exchange solid phase extraction in a fully automated sample treatment procedure. Short- and long-term sample stability data and the analysis of 5000 samples from a clinical trial demonstrate the successful application of our method.