Background: A requirement of current good manufacturing practices for dietary supplements is that manufacturers must identify their dietary ingredients. DNA-based methods can provide species-level authentication that may sometimes be difficult to achieve using conventional morphological and chemical analysis methods. However, because of varying levels of DNA degradation in botanical materials, many commercial tests fail to generate consistent test results across all types of botanical materials. AOAC published guidelines for validation of botanical identification methods and proposed probability of identification (POI) as a method performance parameter. However, few DNA-based botanical authentication methods in the literature follow these guidelines and evaluate POI. Objective: To provide a targeted PCR method validation example that follows AOAC guidelines for validation of botanical identification methods. Methods: Using Matricaria chamomilla (chamomile) as an example, we performed a single-laboratory validation for a targeted PCR method that aimed to identify both raw and processed chamomile materials. The performance parameters of the test were evaluated by carrying out an inclusivity/exclusivity study and a Specified Superior Test Material/Specified Inferior Test Material study to demonstrate that the method's POI meets industry requirements. Results: The chamomile samples were identified by the method and achieved a POI greater than 0.9 with respect to all types of chamomile botanical materials. Conclusions: The method was validated for DNA-based identification of raw and processed chamomile materials, such as sterilized powders and extracts. Highlights: This work will provide insight for laboratories and manufacturers that aim to develop and validate DNA-based botanical identification methods.