The spread and persistence of Listeria monocytogenes in smoked fish products and seafood processing factories are big concerns. Thus, the corresponding quality assurance programs must include adequate microbiological control measures. We evaluated eight different pre-PCR sample processing strategies to be coupled with a previously developed real-time PCR assay for the quantitative detection of L. monocytogenes in salmon products. The optimal pre-PCR procedure involved filtration and DNA purification with the use of a commercial kit. This strategy could detect 10 CFU of L. monocytogenes per g of smoked salmon and could quantify 1,000 CFU/g with excellent accuracy compared with the standard plate count method. Thus, this method could be a promising alternative for the quantitative detection of L. monocytogenes in smoked fish products and processing factories. This method could also detect the bacterium in raw salmon.