To understand the potential human health effects of exposure to hazardous substances among first responders from the East Palestine train derailment, an electronic self-administered Assessment of Chemical Exposures (ACE) survey was created and available to first responders between February-March 2023. Among the 339 completed responder surveys analyzed, most reported working at least 1 day during the incident between February 3-8, 2023. Most (79%) reported inhaling, touching, or swallowing potentially harmful substances and did not report using a face mask or respirator while working (75%). Nearly half reported at least 1 new or worsening physical symptom after incident response. These findings support several recommendations to mitigate exposure to hazardous substances among first responders during future incidents, including using a hierarchy of controls framework to reduce exposure to hazards, timely communication of possible hazardous substances involved in the event, and using the Emergency Responder Health Monitoring Surveillance (ERHMS) framework.