To break the current paradigm in microfluidics that directly links device design to functionality, we introduce microfluidic “virtual channels” that can be dynamically shaped in real-time. A virtual channel refers to a flow path within a microfluidic flow cell, guiding an injected reagent along a user-defined trajectory solely by hydrodynamic forces. Virtual channels dynamically reproduce key microfluidic functionality: directed transport of minute volumes of liquid, splitting, merging and mixing of flows. Virtual channels can be formed directly on standard biological substrates, which we demonstrate by sequential immunodetection at arrays of individual reaction sites on a glass slide and by alternating between local and global processing of surface-adherent cell-block sections. This approach is simple, versatile and generic enough to form the basis of a new class of microfluidic techniques.