We present a novel method for real-time monitoring and kinetic analysis of fluorescence in situ hybridization (FISH). We implement the method using a vertical microfluidic probe containing a microstructure designed for rapid switching between probe solution and nonfluorescent imaging buffer. The FISH signal is monitored in real time during the imaging buffer wash, during which signal associated with unbound probes is removed. We provide a theoretical description of the method as well as a demonstration of its applicability using a model system of centromeric probes (Cen17). We demonstrate the applicability of the method for characterization of FISH kinetics under conditions of varying probe concentration, destabilizing agent (formamide) content, volume exclusion agent (dextran sulfate) content, and ionic strength. We show that our method can be used to investigate the effect of each of these variables and provide insight into processes affecting in situ hybridization, facilitating the design of new assays.