In this study a real-time PCR method has been developed for the specific quantification of the foodborne pathogen Listeria monocytogenes on meat products through the gene hlyA. The PCR was combined with a matrix lysis that allowed the obtaining of the microorganisms without sample dilution and the elimination the PCR inhibitors from dry-cured ham. The qPCR method calibration curve had an efficiency of 100.4%, limits of detection and quantification were 30.1 ± 6.2 CFU/g which is under the legal limit of L. monocytogenes in ready-to-eat products, and an analytical variability <0.25 log hlyA gene copies/reaction. The analysis was performed simultaneously with the reference method ISO 11290-2. The comparison of the qPCR-matrix lysis results with the reference method showed an excellent correspondence, with a relative accuracy between 95.83–105.20%. Finally, the method was applied to commercial derived meat samples and the pathogen was quantified in one of the commercial samples assayed in 69.1 ± 13.9 CFU/g while the reference method did not quantify it. The optimized qPCR showed higher precision and sensitivity than the reference method at low concentrations of the microorganism in a shorter time. Therefore, qPCR-matrix lysis shows a potential application in the meat industry for L. monocytogenes routine control.