In response to environmental stress, there is an increase in the expression of heat shock proteins. Heat shock proteins are molecular chaperones that maintain cell homeostasis through various cellular mechanisms. Heat shock protein 70 is a specific family of heat shock proteins that uses an ATP‐driven mechanism to bind its client proteins. The overexpression of Hsp70 has been shown to prevent apoptosis and lead to tumorigenesis, but the inhibition of Hsp70 has been linked to an increase in apoptosis and tumor cell death. These anti‐apoptotic functions of Hsp‐70 prompted many groups of researchers toward development of sensitive assays for monitoring the ATP‐ase activity of this chaperone which can further be used for discovery of new potential competitive inhibitors of its enzymatic activity. This paper presents the development of resorufin fluorescence based assays for monitoring the ATP‐ase activity of Hsp‐70 by measuring the inorganic phosphate (Pi) or the adenosine diphosphate (ADP) production during the multi turn‐over enzymatic reactions. Enzyme‐coupled reactions were developed to convert the Pi or ADP in hydrogen peroxide which in turn was used to oxidize the reagent amplex red into resorufin which emits fluorescence at 590 nm upon excitation at 530 nm. The non‐hydrolysable ATP analog AMP‐PNP showed inhibition of human Hsp‐70 ATP‐ase activity in the low mM range.