Current proteome profiling techniques have identified relatively few mammalian membrane proteins despite their numerous important functions. To establish a standard throughput‐potential profiling platform for membrane proteins, Triton X‐100‐solubilized rat liver microsomal proteins were separated on a 2‐D separation system (2‐D liquid phase fractionation (PF2D)) in two different pH ranges (4.0–8.5 and 7.0–10.5). This system produced 182 proteins with more than two transmembrane domain (TMD), including 16 TMDs with high confidence. Comparative 2‐D liquid maps with high resolution and reproducibility have been constructed for liver microsome from the phenobarbital (PB) treated rats. PF2D was also found to be useful for the semiquantification of some representative cytochrome P450 family proteins (e.g., cytochrome P450 2B2) that were induced by PB treatment compared with untreated controls. Thus, the combination of both high‐detection capacity and rapid preliminary semiquantification in a PF2D platform could become a standard system for the routine analysis of membrane proteins.