In this work, an electro-kinetic assisted electrospray ionization (EK-ESI) source is proposed and characterized. In EK-ESI, an additional auxiliary electric field is introduced in the liquid flow of a nano-ESI. While traveling forward in the electrospray flow, charged analytes also experience a reverse electric field, which pushes them backwards. As a result, analytes could be separated preliminarily based on their electrophoretic mobility during the electrospray process. Experiments show that EK-ESI can reduce charge competition effects in the ESI source and increase biomolecule detection sensitivities. It was also found that EK-ESI effectively ionizes proteins in a relatively mild solvent condition, which does not require the addition of acids or salt buffers into the solvent. As a proof-of-concept study, a very rough separation effect was observed in this study, further experiments and theoretical study will be carried out to enhance its performances.