A high throughput process including sub-cellular fractionation and multiple protein separation and identification technology allowed us to establish the protein expression profile of Human fetal liver (HFL), which was composed of, at least 2,495 distinct proteins and 568 non-isoform groups identified from 64,960 peptides and 24,454 distinct peptides. In addition to the basic protein identification mentioned above, the MS data were used for complementary identification and novel protein mining. By doing the analysis with integrated protein, EST and genome datasets, 223 proteins and 15 peptides were complementarily identified with high quality MS/MS data.