The ecdysone signaling pathway plays a major role in various developmental transitions in insects. Recent advances in the understanding of ecdysone action have relied to a large extent on the application of molecular genetic tools in Drosophila. Here, we used a comprehensive quantitative SILAC MS-based approach to study the global, dynamic proteome of a Drosophila cell line to investigate how hormonal signals are transduced into specific cellular responses. Global proteome data after ecdysone treatment after various time points were then integrated with transcriptome data. We observed a substantial overlap in terms of affected targets between the dynamic proteome and transcriptome, although there were some clear differences in timing effects. Also, downregulation of several specific mRNAs did not always correlate with downregulation of their corresponding protein counterparts, and in some cases there was no correlation between transcriptome and proteome dynamics whatsoever. In addition, we performed a comprehensive interactome analysis of EcR, the major target of ecdysone. Proteins copurified with EcR include factors involved in transcription, chromatin remodeling, ecdysone signaling, ecdysone biosynthesis, and other signaling pathways. Novel ecdysone-responsive proteins identified in this study might link previously unknown proteins to the ecdysone signaling pathway and might be novel targets for developmental studies. To our knowledge, this is the first time that ecdysone signaling is studied by global quantitative proteomics. All MS data have been deposited in the ProteomeXchange with identifier PXD001455 (http://proteomecentral.proteomexchange.org/dataset/PXD001455).