Hsp70 chaperones take on crucial roles in establishing and maintaining a healthy cellular proteome. Performing a plethora of functions – including supporting protein folding, preventing protein misfolding or aggregation, and resolving protein aggregates – all their activities are reliant on selecting and binding the correct site in a substrate to properly perform the intended function. Research investigating the Hsp70-substrate interaction has mostly been focused on the E. coli Hsp70 DnaK. These studies elucidated a binding preference for hydrophobic sequences often flanked by positively charged residues in substrates with an extended conformation. A stringent selectivity is exhibited for the residue bound in the center of the binding cleft, the anchor residue. Moreover, DnaK was shown to accommodate substrates in two orientations, N-to-C (forward) and C-to-N (reverse). The major cytosolic human Hsp70, Hsc70 (HspA8), shares a great degree of sequence and structure similarity with DnaK. Nonetheless, non-conservative amino acid changes in the immediate substrate binding cleft, and previous studies comparing DnaK and Hsc70 binding sequence selectivity point towards distinct characteristics in their substrate selection. Therefore, this study aimed to further elucidate the binding sequence selectivity and substrate accommodation of Hsc70, in particular, in comparison to that of DnaK, using the model substrate proPhoA. Investigating the binding modes populated by different peptides in Hsc70 through binding affinity measurements, assessment of substrate orientation, and methyl-TROSY NMR of ILV-methyl labeled Hsc70, this study highlights the array of factors influencing substrate accommodation in Hsc70. In many cases, Hsc70 is more promiscuous than DnaK. However, it also appears that residues extending from the anchor residue and a substrate’s backbone/side chain conformation can have significant impact on the chosen binding mode. Most importantly, this project is concluded with a proposed chemical shift fingerprint of I440 in Hsc70’s central binding pocket, reporting on a substrate’s populated binding mode, i.e., the anchor residue identity and binding orientation.