A new online coupling of CIEF with ESI-MS has been developed in glycerol-water media. This improved protocol provides: (i) the electric continuity during the whole analysis by a discontinuous filling of the capillary with 60:40 (cm/cm) catholyte/proteins-ampholyte mixture; (ii) the use of an anticonvective medium, i.e. 30:70 glycerol/water, v/v, compatible with MS detection and as an aid to hydrophobic protein solubilization and (iii) the use of unmodified bare fused-silica capillaries, as the glycerol/water medium strongly reduces EOF. Focusing was performed in positive polarity and cathodic mobilization was achieved by both voltage and pressure application. The setup was optimized with respect to analysis time, sensitivity and precision on pI determination. The optimized anolyte and catholyte were composed of 50 mM formic acid/1 mM glutamic acid (pH 2.35) and 100 mM NH3/1 mM lysine (pH 10.6), respectively. The effects of ampholyte concentration, focusing time and ESI parameters were presented for model proteins and discussed. This new integrated protocol should be an easy and effective additional tool in the field of proteome analysis, providing a means for the characterization of a large number of hydrophilic and hydrophobic proteins.