Nowadays, the importance of dosimetry protocols and formalisms for radiobiology studies is no longer to be demonstrated. Several protocols were proposed for dose determination on low energy X-rays facilities, but depending on irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate. Here, we proposed a new dosimetry protocol for cell irradiations on a low energy X-rays facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer so as close as possible to the real cell irradiation conditions. The different steps of the protocol are the following: i) determination of irradiation parameters (high voltage, intensity, cell container….), ii) determination of the beam quality index (high voltage – half value layer couple), iii) Dose rate measurement with ionization chamber calibrated in air kerma in free in air conditions, iv) quantification of the attenuation of the cell culture medium with EBT3 radiochromic films, v) determination of the dose rate at the cell level. This methodology has to be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of cell monolayer especially considering low energy X-rays.