The use of surfactant mixtures to affect both electroosmotic flow (EOF) and separation selectivity in electrophoresis with poly(dimethylsiloxane) (PDMS) substrates is reported, and capacitively coupled contactless conductivity detection (C4D) is introduced for EOF measurement on PDMS microchips. First, the EOF was measured for two nonionic surfactants (Tween 20 and Triton X-100), mixed ionic/nonionic surfactant systems (SDS/Tween 20 and SDS/Triton X-100), and finally for the first time, mixed zwitterionic/nonionic surfactant systems (TDAPS/Tween 20 and TDAPS/Triton X-100). EOF for the nonionic surfactants decreased with increasing surfactant concentration. The addition of SDS or TDAPS to a nonionic surfactant increased EOF. After establishing the EOF behavior, the separation of model catecholamines was explored to show the impact on separations. Similar analyte resolution with greater peak heights was achieved with mixed surfactant systems containing Tween 20 and TDAPS relative to the single surfactant system. Finally, the detection of catecholamine release from PC12 cells by stimulation with 80 mM K+ was performed to demonstrate the usefulness of mixed surfactant systems to provide resolution of biological compounds in complex samples.