Levels of zinc, along with its mechanistically related metabolites citrate and aspartate, are widely reported as reduced in prostate cancer compared to healthy tissue and are therefore pointed out as potential cancer biomarkers. Previously, it has only been possible to analyze zinc and metabolites by separate detection methods. Through matrix-assisted laser desorption/ionization mass spectrometry imaging (MSI), we were for the first time able to demonstrate, in two different sample sets (n = 45 and n = 4), the simultaneous spatial detection of zinc, in the form of ZnCl3-, together with citrate, aspartate, and N-acetylaspartate on human prostate cancer tissues. The reliability of the ZnCl3- detection was validated by total zinc determination using laser ablation inductively coupled plasma MSI on adjacent serial tissue sections. Zinc, citrate, and aspartate were correlated with each other (range r = 0.46 to 0.74) and showed a significant reduction in cancer compared to non-cancer epithelium (p < 0.05, log2 fold change range: -0.423 to -0.987), while no significant difference between cancer and stroma tissue was found. Simultaneous spatial detection of zinc and its metabolites is not only a valuable tool for analyzing the role of zinc in prostate metabolism but might also provide a fast and simple method to detect zinc, citrate, and aspartate levels as a biomarker signature for prostate cancer diagnostics and prognostics.