The objective of this study was to develop a rapid quantitative detection method by triphosphate (ATP)-bioluminescence and determine the feasibility to assay total bacterial counts (TBC) in probiotic products. A useful pretreatment technique to eliminate the interference to the ATP-bioluminescence method was developed, resulting in a 10–100-fold improvement in the rapid quantitative detection method by ATP-bioluminescence. The TBC obtained by the ATP-bioluminescence rapid detection method was tested against the direct microscopic count method or the plate count method and validated on three ATP Assay Systems. The results generated by the ATP methods and the plate count method were comparable to each other when the interference due to non-microbial cell ATP and matter such as pigment were removed. This study reports the optimisation of an ATP-bioluminescence assay using a pretreatment technique to eliminate interference in order to quickly determine the viable counts of bacteria in solid and liquid probiotic products.