With the completion of genomes of various Aspergillus species, large-scale genome-wide expression studies can be carried out. Genomics, however, is more powerful and efficient when combined with genetics. A multi-copy-based gain-of-function screen is a complementary method to loss-of-function genetic screen and can identify novel genes that may not be easily identifiable through loss-of-function-type screens. Particularly, gain-of-function genetic screens would identify novel activators or repressors of fungal development and secondary metabolism. Here, we describe a working protocol for the identification of novel regulators in Aspergillus nidulans.