In regulatory toxicology, animal tests are habitually applied in hazard assessments to identify the potential toxic endpoints of chemicals to human health and the environment. This paper identifies major challenges associated with the perceptions of the need to rely on animal studies and argues that regulatory culture, resting upon familiarity and confidence in animal studies, hinders the transition to non-animal methodologies (NAMs). These challenges are further compounded by a deferential judicial approach that often fails to uphold the ‘last resort’ principle prescribed by EU chemical legislation. Drawing upon novel empirical data from 32 stakeholder interviews conducted as part of the PrecisionTox project, the study explores the social, technical, and legal barriers to NAM uptake. The findings suggest that progress requires a shift in mindset, improved validation protocols, and proactive legal engagement to replace 20th-century testing methods with 21st-century science.