To understand the relationship of structure to the remarkably diverse bioactivities reported for withanolides, we obtained withaferin A (WA; 1) and 36 analogues (2-37), and compared their cytotoxicity to cytoprotective heat shock-inducing activity (HSA). Analyzing structure-activity relationships for the series, we found that the ring A enone is essential for both bioactivities. Acetylation of 27-OH of 4-epi-WA (28) to 33 enhanced, while introduction of β-OH to WA at C-12 (29) and C-15 (30) decreased both activities. Introduction of β-OAc to 4,27-diacetyl-WA (16) at C-15 (37) decreased HSA without affecting cytotoxicity, but at C-12 (36) it had minimal effect. Importantly, acetylation of 27-OH yielding 15 from 1, 16 from 14, and 35 from 34 enhanced HSA without increasing cytotoxicity. Our findings demonstrate that the withanolide scaffold can be modified to selectively enhance HSA, thereby assisting development of natural product-inspired drugs to combat protein aggregation-associated diseases by stimulating cellular defense mechanisms.