This paper presents parallel microfabrication of three-dimensionally sharp electrospray ionization emitters made out of glass. For the first time, the fabrication of glass emitters relies only on standard microfabrication techniques (i.e., deposition, photolithography, and wet etching), and all manual machining steps are omitted. We also demonstrate a straightforward integration of the three-dimensionally sharp emitter tip with a microfluidic separation channel, which has been one of the major challenges of micro total chemical analysis systems for the past 15 years. As a result, our microfabrication approach provides glass ESI emitters that allow robust performance from run to run and tip to tip and do not suffer from sample spreading at the microchannel outlet. The repeatability of the signal intensity for parallel tips was shown to be within 8.0% RSD (n = 6), and the migration time repeatability for repeated injections was within 6.2% RSD (n = 6). At best, separation plates of up to 2.7 × 10(5)/m were obtained. Since the microfabrication process readily yields three-dimensionally sharp emitter tips, very low ESI voltages (typically 1.4-1.75 kV) suffice for stable ESI, which eventually allows for the use of a variety of different solvent compositions from purely aqueous to high organic content. Here, the advantage of using aqueous conditions is demonstrated in protein analysis.