Activation tagging is a high-throughput method of overexpressing genes by using an enhancer present in insertion sequences that are randomly inserted in the genome to enhance the expression of adjacent genes. Gain-of-function approaches are advantageous to identify the functions of redundant genes that are not identifiable by knockout (KO) mutations, and for identification of phenotypes with small effects, which are enhanced by activation. An activation tag (ATag) library of 800 lines was generated in Arabidopsis ecotype Columbia using the En-I (Spm) transposon system. The ATag lines were used in a forward genetics strategy to identify novel genes that confer resistance/tolerance to abiotic stresses. The ATag lines were screened for altered drought and salt stress response phenotypes using quantitative assays for biomass accumulation under stress, revealing a number of resistant and sensitive ATag mutants.