The Federal Railroad Administration (FRA) recognizes the importance and function of a strong safety culture to railroad safety. Since 2014, FRA’s Office of Research, Development and Technology (RD&T) has supported the American Short Line and Regional Railroad Association (ASLRRA) in its efforts to establish a Short Line Safety Institute (SLSI) focused on improving safety culture on short line and regional railroads. SLSI conducts voluntary, non-punitive, confidential Safety Culture Assessments (SCAs) for short line and regional railroads across the United States. This research summarizes findings from a case study analysis of initial and follow-up SCA reports completed for two railroads.