The blood-brain barrier (BBB) is a major obstacle in treating diseases of the central nervous system (CNS) such as Parkinson's, Alzheimer's, Schizophrenia and brain cancer, affecting 180 million Europeans with less than 5% of current candidate drugs effectively reaching the brain. NAP4DIVE strives to revolutionize the traditionally expensive and inefficient drug development for these diseases by establishing advanced non-animal alternatives for testing and predicting nanoparticle (NP)-based drug delivery across the human BBB. This approach aligns with EU and global initiatives to reduce animal testing and advance human-based biomedical research models. The project will develop two complementary non-animal tools: a high-throughput BBB-on-Chip and an in silico model based on machine-learning (“NP Design Simulator”). A digital repository of optimized nanoparticle designs “NP Design Library” will be created to gather publicly available and newly obtained NP characterisation data, specialised for BBB delivery. The Design simulator screens thousands of NP designs to recommend the most promising ones, which will be tested in vitro on the microfluidic BBB-on-Chip with real-time measurement of barrier integrity. The accuracy and physiological relevance of both tools will be validated by the pharmaceutical partner through comparison with clinical and pre-clinical data. NAP4DIVE tools will reduce animal use in CNS drug development by up to 95% while saving 30 % of costs. By identifying nanoparticles for cross-BBB drug delivery and offering avenues for new effective treatment options, NAP4DIVE addresses one of the most pressing healthcare challenges of the century. A comprehensive HTA will demonstrate market readiness and cost-effectiveness of the tools, an ethical assessment will analyse harm reduction and engagement with regulators and policy makers will promote non-animal alternatives in preclinical testing on a larger scale.