Gain-of-function approaches have been used as an alternative or complementary method to loss-of-function approaches as well as to confer new functions to plants. Gain-of-function is achieved by increasing gene expression levels through the random activation of endogenous genes by transcriptional enhancers or the expression of individual transgenes by transformation. The advantages of gain-of-function approaches compared to loss-of-function approaches for the characterization of gene functions include the abilities to (a) analyze individual gene family members, (b) characterize the function of genes from nonmodel plants using a heterologous expression system, and (c) identify genes that confer stress tolerance to plants that result from the introduction of transgenes. In this review, we describe the current status of gain-of-function mutagenesis and provide several examples of how gene functions have been characterized via high-throughput screening using gain-of-function technology.