This paper describes a fabrication method for the generation of microfluidic structures in glass substrates, based upon microcontact printing and wet chemical etching, with its exemplary application in the field of electrophoretic separation. The manufacturing concept consists of two principle steps. First, a soda-lime glass substrate coated with a chromium/silver layer is masked with hexadecanethiol using an instrument specifically designed for microcontact printing. Second, a series of wet chemical etching steps are performed, resulting in the configuration that was defined by the printer's stamp, being etched into the glass substrate. This process has required the development of specific metallic coatings and the optimisation of etching solutions. For proof of concept, a glass lab-on-a-chip device with a polydimethylsiloxane cover is characterized in terms of electroosmotic flow and is then used for the separation of cystein proteinases from Porphyromonas gingivalis. We anticipate that this approach will be of interest to develop microfluidic glass devices for R&D without expensive microfabrication equipment. This low cost and high throughput process may also be appropriate for mass production.