Bioactive small molecules that show remarkable activities on living organisms always have specific target molecules in the cell. The identification of such target molecules has provided deep insight into drug discovery. We have been aiming to elucidate the mechanism of action of small-molecule compounds by chemical genetics, a new method to discover the interaction between compounds and their intracellular targets, by replacing mutations in genetics with compounds. As a result, we have identified histone deacetylase HDAC, which regulates epigenetics, CRM1, a receptor for protein nuclear export signals, and SF3b, a spliceosome component, all of which are now considered important targets for new anticancer drugs. This approach should be useful not only for the discovery of anticancer targets but also for therapeutic targets for other diseases, such as genetic diseases and intractable rare diseases. In this talk, I will report on the identification of glycolytic enzyme phosphofructokinase and BGLT3, a long non-coding RNA, as the potential targets for the treatment of mitochondrial diseases and sickle cell disease, respectively, based on the latest results of our chemical genetics research.