Pressure pulse injection is proposed as an alternative technique to electrokinetic injection for use in continuous sample flow electrophoresis microchips. During chip operation, sample and buffer solutions are electrokinetically driven near to the entrance of the separation channel in a continuous way. A short sample plug is then injected in the separation channel by a pressure pulse of a few hundred of milliseconds, by mechanically deflecting a soft polymer membrane placed on a dedicated chip reservoir. Our pressure pulse injection technique does not depend on the charge of the analytes and, hence, is characterized by a strongly reduced bias compared to a classical gated flow injection sequence.