Yeast are an ideal system to study Heat Shock Protein 70 (Hsp70) function in a cellular context. This protocol was generated to analyze the function of non-native Hsp70 proteins by expressing them as the sole cytosolic Hsp70 in yeast. As an initial step, Hsp70 variants (such as Ssa1 point mutants and non-yeast versions such as Nematostella vectensis NvHsp70A, B and D) are cloned into an appropriate expression plasmid. Next, these plasmids are transformed into ssa1-4∆ yeast [expressing native Ssa1 from an uracil-based (URA3) plasmid] which are subsequently cured of the original yeast on 5-Fluroorotic Acid (5-FOA). The resulting cells can be screened for a variety of phenotypes which match to the activity of well-studied cellular pathways.