Zebrafish is an excellent and well-recognised organism for investigating vertebrate development. Its proteome is apparently highly similar to that of humans, which makes the zebrafish an attractive model of human diseases. Although genomic and transcriptomic data on zebrafish embryonic development are well established, data on the proteome are scarce, particularly in the cleavage stages. This is mostly due to the high abundance of embryonic yolk proteins, which tend to mask the less abundant cell proteins. In this study, we developed an efficient protocol to reduce the amount of yolk in zebrafish early embryos to improve the shotgun approach-based LC-MS analysis. The analysis of the digested protein samples using the 1D MS/MS shotgun proteomics approach consistently demonstrated that the deyolking procedure resulted in a greater number of total and unique proteins being identified. This protocol resulted in approximately two-fold increase in the number of proteins identified in deyolked samples at cleavage stages. After the maternal-to-zygotic transition, the number of identified proteins increased gradually by 3-4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology analysis revealed a high number of functional proteins in the deyolked samples, such as mitochondrial proteins, while the loss of ribosomal proteins was minimal. This deyolking protocol will improve both qualitative and quantitative proteome analyses, and provides an innovative tool in molecular embryogenesis of yolked animals, such as fish, amphibians, reptiles, and birds. The study was financed by the Research Council of Norway (InnControl project #275786).