The adaptive response of prostate cancer (PCa) cells undergoing androgen targeted therapies (ATT) were investigated. In this study, a long term in vitro ATT model was developed by treating LNCaP PCa cells with AR antagonist Enzalutamide (10 μM) for up to 21 days. Samples were collected at days 7, 14, and 21 of Enzalutamide treatment and compared to vehicle-treated LNCaP cells (DMSO control). High content imaging of fluorescent-labelled lipid probes demonstrate that PCa cells increase lipid uptake and remodel their lipid landscape as a response to ATTs. Interestingly, these ATT-treated cells show little proliferation and mitochondrial activity, suggesting that changes in lipid uptake and content, support pathways other than growth and oxidative phosphorylation. Lipidomics analyses of these cells was undertaken to give a more comprehensive understanding of the changes occurring at the molecular level. Shotgun lipidomics was performed by direct infusion of samples using loop injections on QTRAP 6500 (SCIEX, hybrid triple quadrupole / linear ion trap mass spectrometer). Samples were spiked with internal standards, SPLASH® Lipidomix® (Avanti polar Lipids) and fatty acid C19:0 during lipid extraction. Triple quadrupole functionality was used to perform specific precursor ion (PI) and neutral loss (NL) scans targeting numerous classes of phospholipids, glycerolipids and sterol esters. Data were combined and analysed using LipidViewTM software (SCIEX) providing for lipid identification and quantification of 250 lipids at the sum composition level of identification. In parallel, lipid extracts from all samples were hydrolysed to release fatty acids which were then derivatized to the corresponding methyl ester using a trimethylsulfonium hydroxide reagent and analysed by GC-MS 8040 (Shimadzu 8040, triple quadrupole mass spectrometer). Changes in the absolute abundance of both intact complex lipids and fatty acids will be presented and discussed in the context of cellular responses to ATT.