Diatoms are a major group of microalgae that initiate biofouling by surface colonization of human-made underwater structures; however, the involved regulatory pathways remain uncharacterized. Here, we describe a protocol for identifying and validating regulatory genes involved in the morphology shift of the model diatom species Phaeodactylum tricornutum during surface colonization. We also provide a workflow for characterizing biofouling transformants. By using this protocol, gene targets such as GPCR signaling genes could be identified and manipulated to turn off diatom biofouling. For complete information on the generation and use of this protocol, please refer to Fu et al. (2020).