This paper presents an experimental and numerical investigation into electrokinetic focusing flow injection for bioanalytical applications on 1 x N (i.e., 1 sample inlet port and N outlet ports) and M x N (i.e., M sample inlet ports and N outlet ports) microfluidic chips. A novel device is presented which integrates two important microfluidic phenomena, namely electrokinetic focusing and valveless flow switching within multi-ported microchannels. The study proposes a voltage control model which achieves electrokinetic focusing in a prefocusing sample injection system and which allows the volume of the sample to be controlled. Using the developed methods, the study shows how the sample may be prefocused electrokinetically into a narrow stream prior to being injected continuously into specified outlet ports. The microfluidic chips presented within this paper possess an exciting potential for use in a variety of techniques, including high-throughput chemical analysis, cell fusion, fraction collection, fast sample mixing, and many other applications within the micrototalanalysis systems field.