A rapid test method was developed for detecting mycoplasma contamination in veterinary biological products. The method reduces testing time by 2 weeks and shows comparable sensitivity to the current agar‐based detection model. The primary goals for the development of the test were to reduce the testing time, incorporate a method that was easily adaptable across the veterinary biologics industry and reduce the subjective interpretation of results. We found that biological enrichment is necessary to maintain sensitivity of the detection method when compared to the standard culture‐based test and that periodic sampling of enrichment cultures is essential to detect a wide variety of mycoplasma species that may be present as contaminants. The PCR detection method is comparable to the agar‐based model and can reduce the overall testing time by up to 14 days. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the development of a PCR‐based detection method for the evaluation of veterinary biological products for mycoplasma contamination. The method shows comparability to the current agar detection model and continues to utilize biological enrichment to amplify low levels of mycoplasma and reduce the impact of inhibitory components typically present in veterinary biologics samples.