Peat replacement is an increasing demand in containerized and transplant production, due to the environmental constraints associated to peat use. However, despite the wide information concerning the use of alternative materials as substrates, it is very complex to establish the best materials and mixtures. This work evaluates the use of mixture design and surface response methodology in a peat substitution experiment using two alternative materials (green compost and palm fibre trunk waste) for transplant production of tomato (Lycopersicon esculentum Mill.); melon, (Cucumis melo L.); and lettuce (Lactuca sativa L.) in organic farming conditions. In general, the substrates showed suitable properties for their use in seedling production, showing the best plant response the mixture of 20% green compost, 39% palm fibre and 31% peat. The mixture design and applied response surface methodology has shown to be an useful approach to optimize substrate formulations in peat substitution experiments to standardize plant responses.