Insertional mutagenesis is one of the most effective approaches to determine the function of plant genes. However, due to genetic redundancy, loss-of-function mutations often fail to reveal the function of a member of gene families. Activation tagging is a powerful gain-of-function approach to reveal the functions of genes, especially those with high sequence similarity recalcitrant to loss-of-function genetic analyses. Activation tagging randomly inserts a T-DNA fragment containing engineered four copies of enhancer element into a plant genome to activate transcription of flanking genes. We recently generated a new binary vector, pBASTA-AT2, which has been efficiently used to discover genes involved in BR biosynthesis, metabolism, and signal transduction. Compared to pSKI015, a commonly used activation tagging vector, pBASTA-AT2, contains a smaller size of T-DNA and a bigger number of unique restriction sites within the T-DNA region, making cloning of the flanking sequence a lot easier. Our analysis indicated that pBASTA-AT2 gives dramatically improved transformation efficiency relative to pSKI015. In this article, detailed information about this activation tagging vector and the protocol for its application are provided. Three recommended gene cloning approaches based on the use of pBASTA-AT2, including inverse PCR, thermal asymmetric interlaced PCR, and adaptor ligation-mediated PCR, are described to identify T-DNA insertion sites after selection of activation-tagged mutant plants.