Human heat shock protein 90 (Hsp90) is a molecular chaperone important for the folding, stabilization and quality control of many important client proteins.1 Its role as a hub at the intersection of several fundamental cellular pathways makes it a therapeutic target for pathologies such as neurological disease, cancer and protein folding disorders.2 Hsp90 is also posttranslationally modified at many sites,3 however the structural and functional role of these modifications, individually and in combination, are poorly resolved. Here we use expressed protein selenoester ligation4, 5 to access site-specifically phosphorylated variants of the Hsp90 C-terminal domain, which forms the dimerization interface of the Hsp90 homodimer. The phosphorylations caused only minor conformational changes in the intrinsically-disordered C-terminus, but modulated the chaperone activity. We also extended the semi-synthesis strategy towards Hsp90 variants with modifications in the middle domains. Access to site-specifically modified variants of this large and challenging protein will allow us to elucidate the roles of the posttranslational modifications systematically and investigate their impacts on various client proteins and therapeutics.