The evolution of gene expression programs underlying the development of vertebrates remains poorly characterized. Here, we present a comprehensive proteome atlas of the model chordate Ciona, covering eight developmental stages and 7,000 translated genes, accompanied by a multi-omics analysis of co-evolution with the vertebrate Xenopus. Quantitative proteome comparisons argue against the widely held hourglass model, based solely on transcriptomic profiles, whereby peak conservation is observed during mid-developmental stages. Our analysis reveals maximal divergence at these stages, particularly gastrulation and neurulation. Together, our work provides a valuable resource for evaluating conservation and divergence of multi-omics profiles underlying the diversification of vertebrates.Competing Interest StatementThe authors have declared no competing interest.