Microfluidic devices and methods for using the same are provided. Embodiments include microfluidic devices that have a first separation region configured to separate a sample along a first directional axis based on a first property, and a second separation region in fluid communication with the first separation region and configured to separate the sample along a second directional axis based on a second property. 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.