Sample transport and electrokinetic injection bias are well characterized in capillary electrophoresis and simple microchips, but a thorough understanding of sample transport on devices combining electroosmosis, electrophoresis, and pressure‐driven flow is lacking. In this work, we evaluate the effects of electric fields from 0 to 300 V/cm, electrophoretic mobilities from 10−4 to 10−6 cm2/Vs, and pressure‐driven fluid velocities from 50 to 250 μm/s on sample injection in a microfluidic chemical cytometry device. By studying a continuous sample stream, we find that increasing electric field strength and electrophoretic mobility result in improved injection and that COMSOL simulations accurately predict sample transport. The effects of pressure‐driven fluid velocity on injection are complex, and relative concentration values lie on a surface defined by pressure‐driven flow rates. For high‐mobility analytes, this surface is flat, and sample injection is robust despite fluctuations in flow rate. For lower mobility analytes, the surface becomes steeper, and injection depends strongly on pressure‐driven flow. These results indicate generally that device design must account for analyte characteristics and specifically that this device is suited to high‐mobility analytes. We demonstrate that for a suitable pair of peptides fluctuations in injection volume are correlated; electrokinetic injection bias is minimized; and electrophoretic separation is achieved.