Quantitative Real-Time PCR (qPCR) assays targeting the 16S rDNA was developed as a genus and species specific detection tool for Bifidobacterium and Lactobacillus, and Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus acidophilus LA-5, respectively. Standard curves were established to quantify these probiotic bacteria. The linear regression of standard curves indicated high correlations between the log numbers of pure probiotic culture cells and the Ct values. The assay had a high efficiency and the limit of detection was estimated to be 1.54 ng DNA (corresponding to 104 cells). Results show that qPCR method may be very useful as a rapid, sensitive and specific tool for detecting and quantifying B. animalis subsp. lactis BB-12 and L. acidophilus LA-5 in probiotic supplements. FTIR spectroscopy was used for the first time to determine the ratios of different microorganisms in commercial probiotic supplements. FTIR analysis was also performed for the pure probiotic cultures of B. animalis subsp. lactis BB-12 and L. acidophilus LA-5. Results obtained in this study showed that FTIR spectroscopy is potentially a rapid method for determining probiotic cell components and their ratios in the supplements and verification their detection and identification.