Di(2-pyridyl)ketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di(2-pyridyl)ketone 4--cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) are novel, highly potent and selective anti-tumor and anti-metastatic drugs. Despite their structural similarity, these agents differ in their efficacy and toxicity in-vivo. Considering this, a comparison of their pharmacokinetic and pharmaco/toxico-dynamic properties was conducted to reveal if these factors are involved in their differential activity. Both compounds were administered to Wistar rats intravenously (2 mg/kg) and their metabolism and disposition were studied using UHPLC-MS/MS. The cytotoxicity of both thiosemicarbazones and their metabolites was also examined using MCF-7, HL-60 and HCT116 tumor cells and 3T3 fibroblasts and H9c2 cardiac myoblasts. Their intracellular iron-binding ability was characterized by the Calcein-AM assay and their iron mobilization efficacy was evaluated. In contrast to DpC, Dp44mT undergoes rapid demethylation in-vivo, which may be related to its markedly faster elimination (T = 1.7 h for Dp44mT vs. 10.7 h for DpC) and lower exposure. Incubation of these compounds with cancer cells or cardiac myoblasts did not result in any significant metabolism in-vitro. The metabolism of Dp44mT in-vivo resulted in decreased anti-cancer activity and toxicity. In conclusion, marked differences in the pharmacology of Dp44mT and DpC were observed and highlight the favorable pharmacokinetics of DpC for cancer treatment.