BackgroundInterest in spatial fractionation radiotherapy has exponentially increased over the last decade as a significant reduction of healthy tissue toxicity was observed by mini‐beam irradiation. Published studies, however, mostly use rigid mini‐beam collimators dedicated to their exact experimental arrangement such that changing the setup or testing new mini‐beam collimator configurations becomes challenging and expensive.PurposeIn this work, a versatile, low‐cost mini‐beam collimator was designed and manufactured for pre‐clinical applications with X‐ray beams. The mini‐beam collimator enables variability of the full width at half maximum (FWHM), the center‐to‐center distance (ctc), the peak‐to‐valley dose ratio (PVDR), and the source‐to‐collimator distance (SCD).MethodsThe mini‐beam collimator is an in‐house development, which was constructed of 10 ×  40 mm2 tungsten or brass plates. These metal plates were combined with 3D‐printed plastic plates that can be stacked together in the desired order. A standard X‐ray source was used for the dosimetric characterization of four different configurations of the collimator, including a combination of plastic plates of 0.5, 1, or 2 mm width, assembled with 1 or 2 mm thick metal plates. Irradiations were done at three different SCDs for characterizing the performance of the collimator. For the SCDs closer to the radiation source, the plastic plates were 3D‐printed with a dedicated angle to compensate for the X‐ray beam divergence, making it possible to study ultra‐high dose rates of around 40 Gy/s. All dosimetric quantifications were performed using EBT‐XD films. Additionally, in vitro studies with H460 cells were carried out.ResultsCharacteristic mini‐beam dose distributions were obtained with the developed collimator using a conventional X‐ray source. With the exchangeable 3D‐printed plates, FWHM and ctc from 0.52 to 2.11 mm, and from 1.77 to 4.61 mm were achieved, with uncertainties ranging from 0.01% to 8.98%, respectively. The FWHM and ctc obtained with the EBT‐XD films are in agreement with the design of each mini‐beam collimator configuration. For dose rates in the order of several Gy/min, the highest PVDR of 10.09 ± 1.08 was achieved with a collimator configuration of 0.5 mm thick plastic plates and 2 mm thick metal plates. Exchanging the tungsten plates with the lower‐density metal brass reduced the PVDR by approximately 50%. Also, increasing the dose rate to ultra‐high dose rates was feasible with the mini‐beam collimator, where a PVDR of 24.26 ± 2.10 was achieved. Finally, it was possible to deliver and quantify mini‐beam dose distribution patterns in vitro.ConclusionsWith the developed collimator, we achieved various mini‐beam dose distributions that can be adjusted according to the needs of the user in regards to FWHM, ctc, PVDR and SCD, while accounting for beam divergence. Therefore, the designed mini‐beam collimator may enable low‐cost and versatile pre‐clinical research on mini‐beam irradiation.