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NatureServe, in cooperation with The Nature Conservancy and the U.S. National Park Service, developed this Invasive Species Assessment Protocol as a tool for assessing, categorizing, and listing non-native invasive vascular plants according to their impact on native species and natural biodiversity in a large geographical area such as a nation, state, province, or ecological region. This protocol is designed to make the process of assessing and listing invasive plants objective and systematic, and to incorporate scientific documentation of the information used to determine each species’ rank. NatureServe’s methodology has previously included assessments of the conservation significance of native species; this protocol extends that scope to non-native species as well. The protocol is used to assess species (or infraspecific taxa, as appropriate) individually for a specified “region of interest” and to assign each species an Invasive Species Impact Rank (I-Rank) of High, Medium, Low, or Insignificant to categorize its negative impact on natural biodiversity within that region. The protocol includes 20 questions, each with four scaled responses (A-D, plus U = unknown). The 20 questions are grouped into four sections: Ecological Impact, Current Distribution and Abundance, Trend in Distribution and Abundance, and Management Difficulty. Each species is assessed by considering these questions, with the answers used to calculate a subrank for each of the four sections. An overall I-Rank is then calculated from the subranks. Text comments and citations to information sources should be provided as documentation for each answer selected, along with a concise text summary of the major considerations leading to the overall rank. While designed for use in a specified large, contiguous, biogeographically diverse region, the protocol can be adapted to specified noncontiguous regions (such as the 50 states of the United States), and may also be applied to assess the impact in the non-native range of a species that is also present elsewhere in a region as a native. NatureServe is now using this protocol to assess the biodiversity impact of the approximately 3,500 non-native vascular plant species established outside cultivation in the United States. The protocol is offered here in generalized form for others who might wish to use it to conduct similar assessments and create lists of invasive plants for other nations, states, provinces, ecological regions, or comparable areas.