Prostaglandins (PGs) are important lipid mediators involved in physiological processes, such as inflammation and pregnancy. The pleiotropic effects of the PG isomers and their differential expression from cell types impose the necessity for studying individual isomers locally in tissue to understand the molecular mechanisms. Currently, mass spectrometry (MS)-based analytical workflows for determining the PG isomers typically require homogenization of the sample and a separation method, which results in a loss of spatial information. Here, we describe a method exploiting the cationization of PGs with silver ions for enhanced sensitivity and tandem MS to distinguish the biologically relevant PG isomers PGE2, PGD2, and Δ12-PGD2. The developed method utilizes characteristic product ions in MS3 for training prediction models and is compatible with direct infusion approaches. We discuss insights into the fragmentation pathways of Ag+ cationized PGs during collision-induced dissociation and demonstrate the high accuracy and robustness of the model to predict isomeric compositions of PGs. The developed method is applied to mass spectrometry imaging (MSI) of mouse uterus implantation sites using silver-doped pneumatically assisted nanospray desorption electrospray ionization and indicates localization to the antimesometrial pole and the luminal epithelium of all isomers with different abundances. Overall, we demonstrate, for the first time, isomeric imaging of major PG isomers with a simple method that is compatible with liquid-based extraction MSI methods.