A process for validation is essential in the development of methods that microbial forensics uses to generate reliable and defensible results. Law enforcement investigators need to respond quickly to the best leads to counter ever-increasing threats and will rely upon results generated from the analyses of any microbial forensic evidence to attempt to attribute any attack to a person(s) or group. Readily available technology and knowledge are making it easier for an individual or group to carry out biocrimes or bioterrorism using microorganisms and toxins as weapons. The potential that a biological weapon will be used is of serious concern for the safety and security of people and critical infrastructure. If a biocrime is committed, microbial forensic evidence will be sought, collected, and characterized to help investigators identify the perpetrator(s) and exclude innocent suspects. Analyses of collected material are often challenging because the identification of the signatures most useful for attribution often requires substantial effort (3). In addition, some microbial forensic specimens can be limited in quantity and/or quality. Despite these demands, accurate and credible results are needed because the interpretation of such results might seriously impact the course or focus of an investigation, thus affecting the liberties of individuals, or even be used as a justification for a government’s military response to an attack. Therefore, the methods for the collection, extraction, and analysis of microbial evidence that could generate key results need to be as scientifically robust as possible so that they are defensible to the legal community (12, 21) and, perhaps, to the international government, law enforcement, and scientific communities. Proper interpretation of the results of microbial forensic analysis relies substantially on understanding the performance and limitations of the methods of collection and the analytical processes, assays, and interpretation involved. Failing to properly validate a method or misinterpreting the results from a microbial forensic analysis or process may have severe consequences. DEFINING VALIDATION Validation is frequently used to connote confidence in a test or process. However, frequently, the process of validation is not well defined or properly described in context. Not being explicit about what is meant by validation can result in misinterpretation and misapplication of a properly performed test. It also can lead to a false sense of confidence in a poor method. In the nascent field of microbial forensics (5), there is a need to better describe what constitutes validation. A strict delineation of the steps needed to validate a method or process may be too restrictive; there are a myriad of methods, processes, targets, platforms, and applications. Yet some basic requirements transcend individual differences in methods, and these can be reinforced by contextual description and illustrated with examples. Failing to validate a method or misinterpreting the reliability of a method in a microbial forensic analysis can have dire consequences. This paper provides a framework for developing a validation plan that can be useful for microbial forensics and may have application to other scientific fields where “validation” may be used colloquially.