In a rapidly growing probiotic market, it is essential for probiotic products to meet their label claims of strain content and viable cell counts throughout shelf life to ensure efficacy. DNA based methods are the most commonly used methods for probiotic identification, and plate count methods are the gold standard methods for enumeration. Probiotic health benefits are strain specific. Thus, it would be ideal to use methods that can identify and quantify probiotics at the strain level such as real-time PCR (qPCR) methods. However, developing strain specific qPCR methods can be challenging especially when the target strain belongs to a highly isogenic taxon. Additionally, these methods should be developed and validated according to the validation guidelines including inclusivity, exclusivity, sensitivity, efficiency and accuracy, to ensure reliability of the methods. Here, we describe the process of developing and validating strain specific qPCR methods for probiotic identification and enumeration. Method performance including specificity, efficiency, accuracy and applicability to different product types and product matrices are evaluated. The ability of the enumeration methods to assess strain viability in multi-strain finished products throughout shelf life, and the accuracy of the enumeration methods to estimate the ratios of the target strain in experimental mixtures are assessed. The strain specific qPCR methods demonstrated high specificity, sensitivity, accuracy and applicability. The methods were able to assess strain viability in multi-strain finished products as well as to accurately estimate the ratios of the target strain in experimental mixtures, offering reliable and efficient tools for probiotic product authentication.