Microfluidic methods of assaying molecule switching are provided. Aspects of the methods include microfluidically separating a sample containing the molecule of interest and then employing the resultant separation pattern to determine a switching characteristic of the molecule. Also provided are microfluidic devices, as well as systems and kits that include the devices, which find use in practicing embodiments of the methods. The methods, devices, systems and kits find use in a variety of different applications, such as analytical and diagnostic assays.