The use of mixtures of ionic and zwitterionic surfactants in poly(dimethylsiloxane) (PDMS) microchips is reported. The effect of surfactant concentration on electroosmotic flow (EOF) was studied for a single anionic surfactant (sodium dodecyl sulfate, SDS), a single zwitterionic surfactant (N-tetradecylammonium-N,N-dimethyl-3-ammonio-1-propanesulfonate, TDAPS), and a mixed SDS/TDAPS surfactant system. SDS increased the EOF as reported previously while TDAPS showed an initial increase in EOF followed by a reduction at higher concentrations. When TDAPS was added to a solution containing SDS, the EOF decreased in a concentration-dependent manner. The EOF for all three surfactant systems followed expected pH trends, with increasing EOF at higher pH. The mixed surfactant system allowed tuning of the EOF across a range of pH and concentration conditions. After establishing the EOF behavior, the adsorption/desorption kinetics were measured and showed a slower adsorption/desorption rate for TDAPS than SDS. Finally, the separation and electrochemical detection of model catecholamines in buffer and reduced glutathione in red blood cell lysate using the mixed surfactant system were explored. The mixed surfactant system provided shorter analysis times and/or improved resolution when compared to the single surfactant systems.