α-N-Heterocyclic thiosemicarbazones are among the most promising ribonucleotide reductase inhibitors identified so far. Triapine, the most prominent representative of this class of substances, has been investigated in multiple phase I and II clinical trials. With regard to clinical practice, Triapine showed activity against hematological diseases, but ineffectiveness against a variety of solid tumors. However, the reasons are still vague and the amount of ADME (absorption, distribution, metabolism and excretion) data for Triapine available in the literature is very limited. Therefore, different analytical tools were used to investigate the metabolism of Triapine including electrochemical oxidations, liver microsomes and in vivo samples from mice. The main metabolic reactions, observed by all three methods, were dehydrogenation and hydroxylations, confirming that electrochemistry, as a purely instrumental approach, can be applied for the simulation of metabolic pathways. The dehydrogenated metabolite M1 was identified as a thiadiazole ring-closed oxidation product of Triapine. From a biological point of view, M1, as a key metabolite, is of interest since the crucial chemical property of α-N-heterocyclic thiosemicarbazones to bind metal ions is lost and cytotoxicity studies showed no anticancer activity of M1. The in vivo data of the urine samples revealed very high levels of the metabolites and Triapine itself already 15 min after treatment. This clearly indicates that Triapine is rapidly metabolised and excreted, which represents an important step forward to understand the possible reason for the inefficiency of Triapine against solid tumors.