The development of trans-splicing vectors opens the door for delivering a large therapeutic gene with adeno-associated viral vectors (AAV). One potential application is to deliver the 6 kb mini-dystrophin gene for Duchenne muscular dystrophy (DMD) gene therapy. However, early attempts have been very disappointing because of low transduction efficiency. We have recently identified mRNA accumulation as a critical barrier for the trans-splicing AAV vectors. This barrier can be overcome by rational selection of the gene splitting site. Here we outline a detailed RNase protection assay-based strategy to determine the optimal gene splitting site for the mini-dystrophin gene. We also provide methods to evaluate transduction efficiency of the mini-dystrophin trans-splicing vectors in mdx mouse, a model for DMD.