Cardiolipins are dimeric phospholipids essential for stabilizing mitochondrial structures, regulating the electron transport chain and facilitating apoptosis. Peroxidation and loss of cardiolipins leads to mitochondria dysfunction and cell death, but cancer cells can increase resilience by upregulating cardiolipin synthesis and turnover. We developed an MS approach to quantify cardiolipin turnover using carbon-13 stable isotope. By feeding prostate cancer cell lines with 13C-labelled fatty acids, we demonstrated that these cells consumed media fatty acids to synthesize up to 50% of the cardiolipin species within 6 hours of exposure to the labelled substrates. Among the different acyl-chains, cardiolipins containing linoleic acid (FA 18:2) exhibited the highest turnover rates, compared to palmitic (16:0), stearic (18:0), and oleic (18:1) acids. These fatty acids were incorporated predominantly without modification, meaning without elongation or desaturation. Based on the rate of 13C enrichment, we identified phosphatidylcholine and acyl-carnitine species as key intermediates supporting rapid cardiolipin biosynthesis. Overall, our results highlight cardiolipin biosynthesis as a major cellular sink for exogenous fatty acids, suggesting that the availability of fatty acids in the prostate tumour microenvironment may contribute to cancer growth and survival.