Significant past work has identified unexpected risks of central nervous system (CNS) exposure to the space radiation environment, where long-lasting functional decrements have been associated with multiple ion species delivered at low doses and dose rates. As shielding is the only established intervention capable of limiting exposure to the dangerous radiation fields in space, the recent discovery that pions, emanating from regions of enhanced shielding, can contribute significantly to the total absorbed dose on a deep space mission poses additional concerns. As a prerequisite to biological studies evaluating pion dose equivalents for various CNS exposure scenarios of mice, a careful dosimetric validation study is required. Within our ultimate goal of evaluating the functional consequences of defined pion exposures to CNS functionality, we report in this article the detailed dosimetry of the PiMI pion beam line at the Paul Scherrer Institute, which was developed in support of radiobiological experiments. Beam profiles and contamination of the beam by protons, electrons, positrons and muons were characterized prior to the mice irradiations. The dose to the back and top of the mice was measured using thermoluminescent dosimeters (TLD) and optically simulated luminescence (OSL) to cross-validate the dosimetry results. Geant4 Monte Carlo simulations of radiation exposure of a mouse phantom in water by charged pions were also performed to quantify the difference between the absorbed dose from the OSL and TLD and the absorbed dose to water, using a simple model of the mouse brain. The absorbed dose measured by the OSL dosimeters and TLDs agreed within 5–10%. A 30% difference between the measured absorbed dose and the dose calculated by Geant4 in the dosimeters was obtained, probably due to the approximated Monte Carlo configuration compared to the experiment. A difference of 15–20% between the calculated absorbed dose to water at a 5 mm depth and in the passive dosimeters was obtained, suggesting the need for a correction factor of the measured dose to obtain the absorbed dose in the mouse brain. Finally, based on the comparison of the experimental data and the Monte Carlo calculations, we consider the dose measurement to be accurate to within 15-20%.