Recent Food Safety Modernization Act (FSMA) provisions for the protection of public health allow growers to use agricultural water exceeding the quantitative standards for indicator Escherichia coli if a pre-harvest waiting period after the last irrigation is applied, for a calculated number of days based on an assumed microbial die-off rate. There is an urgent need to validate the microbial die-off rate under standardized, multi-commodity and multi-regional conditions. Our objective is to establish die-off rates of a standardized set of indicators and attenuated pathogens on baby-spinach and -lettuce in a replicated trial under field conditions in three different climatic regions. The estimated die-off rate, and the identified factors that affect it, will provide the foundation for a validated produce- and region-specific agricultural water die-off matrix for stakeholders to more effectively assess water quality and use. Our second objective is to develop a mathematical model of pathogen die-off under relevant environmental conditions and industry practices. The model will predict pathogen die-off under diverse conditions and indicate the conditions under which the regulatory matrix for pathogen die-off in agricultural water may be relied upon. Collectively, the results are expected to strengthen the FSMA regulatory matrix or demonstrate the need to modify it.