BACKGROUND AND PURPOSE: Image-guided equipment for precision irradiation of small animals for pre-clinical radiotherapy research became recently available. To enable downscaled radiotherapy studies that can be translated into human radiotherapy knowledge, a treatment planning system for pre-clinical studies is required. MATERIAL AND METHODS: A dedicated treatment planning system (SmART-Plan) for small animal radiotherapy studies was developed. It is based on Monte Carlo simulation of particle transport in an animal. The voxel geometry is derived from the onboard cone beam CT imaging panel. SmART-Plan was validated using radiochromic film (RCF) dosimetry in various phantoms: uniform, multislab and a realistic plasticized mouse geometry. RESULTS: Good agreement was obtained between SmART-Plan dose calculations and RCF dose measurements in all phantoms. For various delivered plans agreement was obtained within 10% for the majority of the targeted dose region, with larger differences between 10% and 20% near the penumbra regions and for the smallest 1mm collimator. Absolute depth and lateral dose distributions showed better agreement for 5 and 15-mm collimators than for a 1-mm collimator, indicating that accurate dose prediction for the smallest field sizes is difficult. CONCLUSION: SmART-Plan offers a useful dose calculation tool for pre-clinical small animal irradiation studies.