The charged linker region in Hsp90 has been shown to be important for Hsp90 function and global conformation. In humans, two serines located in the charged linker region are constitutively phosphorylated in vivo. In order to gain knowledge on the possible effect of these phosphorylation sites on Hsp90 global conformation, we purified a human Hsp90β mutant where both serines are mutated to alanines, and used limited proteolysis to study the “proteolytic” fingerprint of our mutant and the wild-type, phosphorylated Hsp90β. We observed that serine to alanine mutation resulted in increased tryptic cleavage in the C-domain of Hsp90β.