Measuring binding affinities and association/dissociation rates of molecular interactions is important for a quantitative understanding of cellular mechanisms. Many low-throughput methods have been developed throughout the years to obtain these parameters. Acquiring data with higher accuracy and throughput is, however, necessary to characterize complex biological networks. Here, we provide an overview of a high-throughput microfluidic method based on mechanically induced trapping of molecular interactions (MITOMI). MITOMI can be used to obtain affinity constants and kinetic rates of hundreds of protein-ligand interactions in parallel. It has been used in dozens of studies to measure binding affinities of transcription factors, map protein interaction networks, identify pharmacological inhibitors, and perform high-throughput, low-cost molecular diagnostics. This article covers the technological aspects of MITOMI and its applications.