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Simple Summary: Cell killing and tumor response in cancer patients depends not only on the absorbed radiation dose but also on the dose rate and delivery time. In this study, a biodosimetry assay based on the frequency of dicentrics chromosomes scored in peripheral blood lymphocytes from prostate cancer patients and PC3 human prostate cancer cell line was used to investigate the radiobiological impact of the relative prolonged dose delivery time and/or decreased dose rate met in advanced modulated radiotherapy techniques (VMAT and IMRT) compared to conventional non-modulated (3D-CRT) in prostate patient plan irradiations. The results showed a small but statistically significant decrease in the number of dicentrics following radiation with the modulated techniques, suggesting a corresponding decrease on the radiation dose efficiency. The biodosimetry assay could be used as an alternative to the laborious conventional clonogenic assay, while both lymphocytes and cancer cell line could effectively be used for estimation of the biological absorbed dose. While rapid technological advances in radiotherapy techniques have led to a more precise delivery of radiation dose and to a decreased risk of side effects, there is still a need to evaluate the efficacy of the new techniques estimating the biological dose and to investigate the radiobiological impact of the protracted radiotherapy treatment duration. The aim of this study is to compare, at a cytogenetic level, advanced radiotherapy techniques VMAT and IMRT with the conventional 3D-CRT, using biological dosimetry. A dicentric biodosimetry assay based on the frequency of dicentrics chromosomes scored in peripheral blood lymphocytes from prostate cancer patients and PC3 human prostate cancer cell line was used. For each patient blood sample and each subpopulation of the cultured cell line, three different irradiations were performed using the 3D-CRT, IMRT, and VMAT technique. The absorbed dose was estimated with the biodosimetry method based on the induced dicentric chromosomes. The results showed a statistically significant underestimation of the biological absorbed dose of ~6% for the IMRT and VMAT compared to 3D-CRT irradiations for peripheral blood lymphocytes, whereas IMRT and VMAT results were comparable without a statistically significant difference, although slightly lower values were observed for VMAT compared to IMRT irradiation. Similar results were obtained using the PC3 cell line. The observed biological dose underestimation could be associated with the relative decreased dose rate and increase irradiation time met in modulated techniques compared to the conventional 3D-CRT irradiations. [ABSTRACT FROM AUTHOR]