The exact mechanisms leading to alternative splice site selection are still poorly understood. However, recently cotransfection studies in eukaryotic cells were successfully used to decipher contributions of RNA elements (cis-factors), their interacting protein components (trans-factors) or the cell type to alternative pre-mRNA splicing. Splice factors often work in a concentration dependent manner, resulting in a gradual change of alternative splicing patterns of a minigene when the amount of a trans-acting protein is increased by cotransfections. Here, we give a detailed description of this technique that allows analysis of large gene fragments (up to 10-12 kb) under in vivo condition. Furthermore, we provide a summary of 44 genes currently investigated to demonstrate the general feasibility of this technique.