The model organism, zebrafish (danio rerio), is particularly amenable to studies deciphering regulatory genetic networks in vertebrate development, biology and pharmacology. Unraveling the functional dynamics of such networks requires precise quantitation of protein expression during organismal growth, which is incrementally challenging with progressive complexity of the systems. In an approach towards such quantitative studies of dynamic network behavior, we have applied mass spectrometric methodology and rigorous statistical analysis to create comprehensive, high-quality profiles of proteins expressed at two stages of zebrafish development. Proteins of embryos 72 and 120 hours post fertilizations (hpf) were isolated and analyzed both by two dimensional (2D) liquid chromatography (LC) followed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and by 2D polyacrylamide gel electrophoresis (PAGE) followed by matrix-assisted laser desorption ionization tandem time-of-flight (MALDI-TOF/TOF) protein identification. We detected 1384 proteins from 327,906 peptide sequence identifications at 72 hpf and 120 hpf with false identification rates of less than 1% using 2D-LC-ESI-MS/MS. These included only approximately 30% of proteins that were identified by 2D-PAGE-MALDI-TOF/TOF. Roughly 15% of all detected proteins were derived from hypothetical or predicted gene models or were entirely un-annotated. Comparison of proteins expression by 2D differential in gel electrophoresis (DIGE) revealed that proteins involved in energy production and transcription / translation were relatively more abundant at 72 hpf consistent with faster synthesis of cellular proteins during organismal growth at this time compared with 120 hpf. The data are accessible in a database that links protein identifications to existing resources including the Zebrafish Information Network data base. This new resource should facilitate the selection of candidate proteins for targeted quantitation and refine systematic genetic network analysis in vertebrate development and biology.