Glycogen Synthase Kinase 3 beta (GSK3b) is a serine/threonine protein kinase, which is part of the PI3Kinase/Akt-, Wnt- and Hedgehog-pathway and is involved in the regulation of gene transcription, protein synthesis and apoptosis. GSK3b is implicated in the development of cardiac hypertrophy, type 2 diabetes and Alzheimers' disease. To systematically analyze GSK3b dependent signal transduction we aimed to identify GSK3b associated protein complexes via Tandem Affinity Purification (TAP) and fused a TAP-Tag consisting of a Flag/Strep II Tag at the C-terminal end. This enables us to perform a two-step purification of the recombinant GSK3b and its interacting partners under native conditions. We found a similar behaviour of the recombinant GSK3b -TAP-Tag compared to the endogenous GSK3b concerning subcellular localisation, phosphorylation and enzymatic activity. Gel filtration analyses showed that both enzymes, GSK3b and GSK3b-TAP-Tag, formed high molecular weight complexes in HEK293 cells. MS-based analysis of copurified proteins revealed that Axin, ATP-citrate synthase and Acetyl-CoA-carboxylase, which are known to be substrates of GSK3b, form stable complexes with GSK3b. In the case of Acetyl-CoA-carboxylase we validated the complex formation by immunoprecipitation. Moreover we identified new interaction partners e.g. Alanyl-tRNA synthetase, Annexin A2, Elongation factor 1, Filamin, Glyceraldehyde-3-phosphate dehydrogenase, Heat shock cognate 71 protein, Nuclear migration protein nudC. Conclusion: TAP is a powerful method to identify interacting proteins of GSK3b. Several known interacting enzymes of GSK3b were identified, but also new interacting partners of GSK3b were found. Therefore the TAP approach may helps to elucidate novel functions of GSK3b in cellular processes such as cell metabolism, cytoskeletal organization and subcellular transport.