Herein, we present a straightforward surface modification technique for PDMS-based microfluidic devices. The method takes advantage of the high reactivity of concentrated sulfuric acid to enhance the surface properties of PDMS bulk material. This results in alteration of the surface morphology and chemical composition that is in-depth characterized by ATR-FTIR, EDX, SEM, and XPS. In comparison to untreated PDMS, modified substrates exhibit a significantly reduced diffusive uptake of small organic molecules while retaining its low electroosmotic properties. This was demonstrated by exposing the channels of a microfluidic device to concentrated rhodamine B solution followed by fluorescence microscopy. The surface modification procedure was used to improve chip-based electrophoretic separations. Separation efficiencies of FITC-labeled amines/amino acids obtained in treated and untreated PDMS-devices as well as in glass chips were compared. We obtained higher efficiencies in H2SO4 treated PDMS chips compared to untreated ones but lower efficiencies than those obtained in commercial microfluidic glass devices.