The current report discusses the process in which a methods comparison study in the National Animal Health Laboratory Network is performed. Specific details are provided for designing and analyzing studies intended to evaluate analytical sensitivity, efficiency, analytical specificity, cross-contamination, repeatability, operator variability, and to compare the performance of methods using diagnostic samples. As an example, a case study is presented comparing the performance of a candidate reverse transcription polymerase chain reaction (RT-PCR) chemistry to the current RT-PCR chemistry in use when the assay was originally validated. The present study revealed that, for all of the validation factors evaluated, the candidate method performed at least as well and generally better than the current method. The candidate method was, therefore, deemed fit for the original intended purpose of the current method and rendered acceptable for use. A discussion of the case study is intended to further motivate consideration of the study designs chosen.