Amniotic fluid is a dynamic and complex mixture that reflects the physiological status of the developing fetus. In this study, the human amniotic fluid (AF) proteome of a 16-18 week normal pregnancy was profiled and analyzed, to investigate the composition and functions of this fluid. Due to the complexity of AF, we utilized three different fractionation strategies, to provide greater coverage. Two types of two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC/MS/MS) as well as an LC-SDS-PAGE-LC-MS/MS platform were used. A total of sixteen AF samples between gestational ages of 16 to 18 weeks, from women carrying chromosomally normal fetuses were analyzed by one of the three fractionation methods, followed by a common reverse phase-LC-MS/MS step. Mascot and GPM engines were used to search the IPI human database for peptide sequence identification. The list of proteins was generated by combining the results of both engines, through the Peptide Prophet of Scaffold software. All identified proteins were combined to generate the AF proteome, comprising 1,026 unique gene matches or 842 non-redundant proteins. This list includes most of the currently used biomarkers for pregnancy-associated pathologic conditions such as preterm delivery, intra-amniotic infection and chromosomal anomalies of the fetus. The subcellular localization, tissue expression, functions and networks of the AF proteome were analyzed by various bioinformatic tools. These data will contribute to the better understanding of amniotic fluid function and to the discovery of novel biomarkers for prenatal diagnosis of fetal abnormalities.