Lipid metabolism is exquisitely sensitive to androgen signalling in prostate cancer cells and cellular lipid composition is significantly altered during prostate cancer development. Here we studied the lipidome of human prostate tumours, to determine whether alterations in cellular phospholipids are directly androgen regulated and are associated with clinical features of the tumours. Using ex vivo culture of human prostate tumours, we generated a cohort of matched explants from >90 patients, cultured in the presence or absence of the current androgen receptor antagonist enzalutamide. Lipids extracted from whole tumour homogenates were analysed for all major phospholipid species by electrospray ionisation tandem mass spectrometry. Analysis of the cohort revealed characteristic enzalutamide-induced alterations in fatty acid chain elongation and saturation, and identified individual lipid species whose change in abundance was associated with an anti-proliferative response to the drug. As there was significant inter-patient heterogeneity observed in the lipid profiles and response to enzalutamide, we utilised Matrix Assisted Laser Desorption/Ionisation (MALDI) imaging coupled with tissue histology to view the spatial distribution of distinct phospholipid species within discrete pathological regions of these tissues, enhancing the identification of lipid and metabolite alterations. Moreover, we directly imaged the distribution of enzalutamide throughout the tissue and showed that, when dissolved in the tissue culture media, the drug completely penetrates the tissue core within 2 hrs of culture. In summary, MALDI mass spectrometry imaging of human prostate tumour explants has not only provided spatial information on drug distribution not previously achievable, it has yielded cell type-specific profiles of lipids and their altered abundance in response to androgenic targeting.