AbstractBasophils represent the rarest type of granulocyte in human peripheral blood. Thus, researching basophils has historically been challenging and has often been reliant on enrichment protocols using density gradient centrifugation. This article describes a novel, fast, and cost‐effective method to purify highly viable human basophils from peripheral blood through negative immunomagnetic selection, foregoing the density centrifugation step in the Basic Protocol. The technique is easy to use and consistently produces purities >96%. Furthermore, the Support Protocols describe procedures to determine basophil yield, purity, and viability, and how to investigate functional activity of the purified basophils through flow cytometry and visualize the basophils through microscopy. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol: Gradient centrifugation‐independent basophil isolationSupport Protocol 1: Flow cytometry staining to assess basophil yield, purity, and viabilitySupport Protocol 2: Giemsa stainingSupport Protocol 3: Calcium flux analysisSupport Protocol 4: Basophil activation test