Alveolar rhabdomyosarcoma (RMA) is a highly malignant soft tissue tumor in children with poor prognosis and failure of established therapies in advanced stages. Therefore, novel treatment options are required. Photodynamic therapy (PDT) has been found useful for the treatment of different tumor entities and might represent such a novel treatment option. A major limitation of PDT remains the restriction to superficial tumor cell layers as illumination with light is essential for the generation of reactive oxygen species. Current research focusses on the development of modified Hypericin (HYP)-based photosensitizers, as well as combining PDT and targeted internal radiotherapy with I, to generate an additive anti-tumor effect.A standardized protocol for in vitro Hypericin-PDT was established in RMA cells. The anti-tumor properties of this photosensitizer were analyzed on molecular and metabolic levels. Changes in cell morphology were visualized using bright field-, fluorescence- and scanning-electron microscopy. Iodinated Hypericin derivatives with both radioactive and non-radioactive isotopes I/I were employed to establish a targeted radionuclide therapy and investigate the potential of a combined treatment with PDT.In vitro photodynamic treatment with Hypericin showed a strong anti-tumor efficiency with favorable cellular uptake and compromised cancer cells on metabolic and molecular levels. Iodination of the photosensitizer did not impair the photosensitizer´s properties. Targeted radiotherapy with I-HYP led to distinct reductions of tumor viability. A simultaneously performed PDT leads to a reduction of cell viability that begins earlier in time. However, an additive enhancement of the cell viability was not observed in the selected dose range.In this in vitro study, we got a first insight of a possible potential of Hypericin for the treatment of pediatric soft tissue sarcoma. By coupling with radioiodine, we developed a novel approach for a combined anti-tumor treatment. The in vitro experiments lay the foundation for further in vivo experiments, which are needed to study the effects of a sequential administration of I-HYP and HYP.