The Apicomplexan parasite Toxoplasma gondii is an obligate intracellular parasite. Infection by T .gondii causes the disease toxoplasmosis, which is one of the most prevalent parasitic diseases of animals and humans. It has been 100 years since the first discovery of the parasite in 1908; research on T. gondii has been carried out in many scientific disciplines consistently expanding the understanding of this parasite. In the last ten years, the developments of EST, microarray, genome sequencing and continuing efforts towards genome annotation has centralized the focus of T. gondii research on the understanding of gene expression and gene functions on the genome scale. Equipped with the technical advances in mass spectrometry and bioinformatics, proteomics has become established as an integral component in the post-genomics era by providing first-hand data on the functional products of gene expression. In this study, three complementary proteomic strategies, 1-DE, 2-DE and MudPIT, have been used to characterise the proteome of T. gondii tachyzoites. Protein identifications have been acquired for more than two thousand (2252) unique release 4 genes, representing almost one third (29%) of the predicted proteome of all life cycle stages. Functional predictions for each protein were carried out, which provided valuable insights into the composition of the expressed proteome and their potential biological roles. The T. gondii proteomic data has been integrated into the publically accessible ToxoDB, where 2477 intron-spanning peptides provided supporting evidence for correct splice site annotation of the release 4 genome annotation. The incompleteness of the release 4 genome annotation has been highlighted using peptide evidence, confirming 421 splice sites that are only predicted by alternative gene models. Analysis has also been carried out on the proteomic data in the light of other genome wide expression data. The comparison of the proteome and transcriptome of Toxoplasma and other Apicomplexa parasites has revealed important discrepancies between protein and mRNA expression where interesting candidates have been highlighted for further investigation. A preliminary DIGE study has been developed to characterize protein expression changes in T. gondii grown in the presence or absence of glucose. In conclusion, this study has demonstrated the importance of proteomic applications in understanding gene expression profiles and regulation in T. gondii and highlighted the importance and potential of proteogenomic approaches in genome annotation process. The importance of temporal and quantitative proteomics as well as the future of systems biology has been discussed.