A recent study illustrated that a fluorescence polarization assay can be used to identify substrate-competitive Hsp70 inhibitors that can be isoform-selective. Herein, we use that assay in a moderate-throughput screen and report the discovery of a druglike amino-acid-based inhibitor with reasonable specificity for the endoplasmic reticular Hsp70, Grp78. Using traditional medicinal chemistry approaches, the potency and selectivity were further optimized through structure-activity relationship (SAR) studies in parallel assays for six of the human Hsp70 isoforms. The top compounds were all tested against a panel of cancer cell lines and disappointingly showed little effect. The top-performing compound, 8, was retested using a series of endoplasmic reticulum (ER) stress-inducing agents and found to synergize with these agents. Finally, 8 was tested in a spheroid tumor model and found to be more potent than in two-dimensional models. The optimized Grp78 inhibitors are the first reported isoform-selective small-molecule-competitive inhibitors of an Hsp70-substrate interaction.