Hsp90, as a key molecular chaperone, plays an important role in modulating the activity of many cell signaling proteins and is an attractive target for anticancer therapeutics.Herein, we report the discovery of N-pyridoyl-Δ 2 -pyrazoline analogs as novel Hsp90 inhibitors by integrated approaches of drug design, organic synthesis, cell biology, and qualitative proteomic analysis. Novel chemical compounds were designed and optimized in the adenosine triphosphate-binding site of Hsp90; lead optimized compounds were found to have significant interactions with Asp93 and other amino acids crucial for Hsp90 inhibition. The designed compounds were synthesized by a two-step procedure; different aromatic aldehydes were reacted with various acetophenones to form substituted 1,3-diphenyl-prop-2-enones (Ic-Io), which upon reaction with isonicotinic acid hydrazide in the presence of glacial acetic acid form N-pyridoyl-Δ 2 -pyrazoline compounds (PY1-PY13). Compounds PY3, PY2, and PY1 were identified as potential leads amongst the series, with promising anticancer activity against human breast cancer and melanoma cells, and the ability to inhibit Hsp90 similar to radicicol by drug-affinity responsive target stability proteomic analysis in a whole-cell assay.