Background: Despite the advent of chemotherapy and androgen-receptor signaling inhibitors (ARSi), patients with metastatic castration-resistant prostate cancer (mCRPC) still show poor prognosis and reduced survival. The selective pressure induced by treatments favors the activation of alternative metabolic pathways that allow the persistence of cancer cells in unfavorable conditions. This study aimed at assessing the lipidomic profiles of patients with mCRPC, in order to identify lipid species potentially useful to predict for prognosis and response to therapies. Methods: Plasma samples were collected from patients with mCRPC who were starting a first-line treatment for mCRPC (1L) (n = 30) and from those who had already received at least two lines of treatment for mCRPC ( > 2L) (n = 19), including at least one ARSi and a taxane. Lipids were extracted from plasma samples using a modified Matyash method employing a mix of cold MeOH and MTBE and containing a mix of deuterated standards (Splash Lipidomix). Lipids were then analyzed with an untargeted lipidomic approach using an UHPLC Vanquish system coupled with a Q-Exactive Plus instrument. T-test was then applied to identify lipid species and classes that were differentially expressed in 1L vs < 2L patients. Results: We identified and quantified a total of 907 plasma lipids. Overall, 68 lipid species were significantly dysregulated in > 2L compared to 1L plasma samples. 56 species were found to be upregulated, whereas 12 were downregulated, with a fold change (FC) > 1.3 or < 0.75 and a p-value < 0.05. At the level of lipid classes, > 2L patients showed higher levels of acylcarnitine (CAR), diacylglycerols (DG), phosphatidylethanolamine (PE) and triacylglycerols (TG). The following lipids showed the highest FC: DG28:2 = 4.2; CAR14:0 = 3.7; CAR20:1 = 2.6; CAR18:0 = 2.3; PE40:6 = 2.3. Conversely, significantly lower levels of specific phosphatidylcholines (PC) and sterols (ST) were found in > 2L compared to 1L patients. The following species showed the lowest FC: PCO-39:3 = 0.52; ST29:1;O;S = 0.55; PC36:5 = 0.55; PC36.4 = 0.60. These results suggest that patients with strongly pretreated mCRPC show higher levels of lipid species involved in the switch between the glucose and fatty acid metabolism. We also found a dysregulation of ceramides and sphingomyelins which are well-known lipid species involved in apoptosis and cancer progression. Specific lipids warrant further investigation to be used as potential prognostic and predictive biomarkers in patients with mCRPC. Conclusions: Using a quantitative mass spectrometry approach, we investigated the dysregulation of lipids and lipid metabolism in mCRPC patients at different disease stages. We found that specific lipid species show differential levels in patients pretreated with ARSi and chemotherapy, compared to those who are naïve to these treatments, paving the way for further investigations in this field.