This study reports an approach to dynamically separate cultured human B-lymphoma (Raji) cells from normal human red blood cells (RBCs), mixed in an isotonic solution, by utilizing a hydrogenated amorphous silicon-based optically-induced electrokinetics chip. A theoretical calculation of the polarization model for the two different types of cells is first presented. The differential velocity between the cell types, generated as a result of the optically-induced dielectrophoresis (ODEP) force is obtained using a finite element method. The theoretical results indicate that the two types of cells could be separated by using the ODEP force. Experimental results verified that the two types of cells can be separated by using two optically projected lines of different widths, with one projected line moving and the other line stationary, as the virtual electrodes for generating the ODEP force. Separation is achieved with these virtual electrodes using a bias potential of 20 V pp at a driving frequency of 50 kHz. To the best of our knowledge, this work is the first experimental validation of rapid separation of Raji cells from RBCs using optically-induced electrokinetics. © 2013 IEEE.