The risk assessment of genetically modified plants in the European Union includes the evaluation of newly expressed (novel) proteins with regards to their safety for humans, animals and the environment. In applications for food and feed uses, toxicity and allergenicity for humans and animals represent the main areas of possible safety concerns associated to newly expressed proteins; the very limited exposure (spillage) would preclude environmental issues. Dedicated tools and methodologies for the assessment of newly expressed proteins in genetically modified plants are thoroughly described in EFSA guidance documents and sectoral legislation, and an overview will be provided. In this context, pore forming proteins (e.g. Cry proteins) newly expressed in genetically modified plants are regularly assessed by EFSA, and examples will be presented. Noteworthy, the safety assessment of novel proteins, including pore forming proteins, is set on the paradigm for the risk assessment of chemicals and uses related tools. These are based on animal studies and are not fully fitting the purpose, considering the nature and complexity of proteins as compared to small molecules, requiring adaptation. Nowadays the scientific developments in protein science and related high-quality publicly available information offer the opportunity to strengthen and modernise the safety assessment of novel proteins, focusing on a mechanistic understanding of toxicity in a 3Rs perspective. EFSA developments on in silico and in vitro tools for novel protein safety assessment will be presented.