Synchrotron radiation is an excellent tool for investigating bystander effects in cell and animal models because of the well-defined and controllable configuration of the beam. Although synchrotron radiation has many advantages for such studies compared to conventional radiation, the contribution of dose exposure from scattered radiation nevertheless remains a source of concern. Therefore, the influence of scattered radiation on the detection of bystander effects induced by synchrotron radiation in biological in vitro models was evaluated. Radiochromic XRQA2 film-based dosimetry was employed to measure the absorbed dose of scattered radiation in cultured cells at various distances from a field exposed to microbeam radiotherapy and broadbeam X-ray radiation. The level of scattered radiation was dependent on the distance, dose in the target zone and beam mode. The number of gamma-H2AX foci in cells positioned at the same target distances was measured and used as a biodosimeter to evaluate the absorbed dose. A correlation of absorbed dose values measured by the physical and biological methods was identified. The gamma-H2AX assay successfully quantitated the scattered radiation in the range starting from 10 mGy and its contribution to the observed radiation-induced bystander effect.