Autonomous Vehicles (AVs) will transform road safety and efficiency in the years to come, but achieving this requires large-scale deployment, trust, and understanding from all human road users, including drivers and bicyclists. External Human-Machine Interfaces (eHMIs) are becoming a crucial part of the process, enabling intuitive communication between AVs and other road users. This project aims to develop, assess, and optimize the concept of eHMIs to foster positive perceptions, build trust, and ensure safe interactions in mixed traffic scenarios. This study will involve a test of about 40 participants who will interact with AVs fitted with various eHMI prototypes under controlled conditions using driving and bicycle simulators. Behavioral metrics like the perception-reaction time (PRT), the perceived level of comfort, and the perceived level of trust, as well as transportation metrics like travel time, intersection clearance time, and near-miss incidents, will be assessed for different designs for the eHMI, including visual-based (LED Displays, Symbolic Messages, Color-coded Signals, Animated Indicators, etc.) and multimodal designs. Longitudinal experiments will measure the impact of acclimatization and determine the best eHMI setups, followed by field tests under realistic conditions for verification. User-focused optimization tools will also be designed to adapt enhanced eHMI setups to various demands and scenarios. Expected outcomes will include best-in-class eHMI designs for increased road safety, operational efficiency, and user confidence, providing valuable guidance for city planners, policymakers, and AV manufacturers.