Tumor directed cytotoxic therapy is one of the major challenges for the success of chemotherapy. In order to accomplish this goal in neuroblastoma, we rationally designed a prodrug of etoposide as substrate for tyrosine hydroxylase, a well established neuroblastoma associated enzyme. Here, we report synthesis and characterization of a 3,4 dihydroxy-phenyl carbamate derivative of etoposide. In order to demonstrate activation by tyrosine hydroxylase, the coding sequence of murine tyrosine hydroxylase was generated by reverse transcriptase-polymerase chain reaction from NXS2 neuroblastoma cells and cloned into the pRSET-A bacterial expression vector. The enzyme was expressed in Escherichia coli, characterized by Western blot and enzymatic activity was demonstrated by conversion of tyrosine into DOPA in the presence of cofactors using reversed phase high-performance liquid chromatography. Under these enzymatic conditions, we demonstrate conversion of 3,4 dihydroxy-phenyl carbamate prodrug into free etoposide. This effect was clearly mediated by the enzyme since bacteria transformed with the empty vector were ineffective of prodrug activation. Furthermore, tyrosine hydroxylase positive cells exposed to the etoposide prodrug were effectively killed in contrast to tyrosine hydroxylase negative controls. These findings demonstrate that etoposide can be designed as a prodrug substrate for tyrosine hydroxylase and thereby establish proof of concept for neuroblastoma directed enzyme prodrug therapy.