Multi-directional microfluidic devices and methods for using the same are provided. Aspects of the invention include microfluidic devices that are configured to subject a sample to two or more directionally distinct flow fields, and include a separation medium and a binding medium, where the binding medium is in fluid communication with the separation medium. Also provided are methods of using the devices as well as systems and kits that include the devices. The devices, systems and methods find use in a variety of different applications, including diagnostic and validation assays.