The microbiological standard for detection of Listeria monocytogenes relies on several cultural steps and requires 7 days for final confirmation, and due to food distribution and market demands, there is a prevailing need for an alternative methodology for its detection. The aim of this study was to compare different detection strategies based on real-time PCR (RTi-PCR) for a rapid and sensitive detection in an ample range of food products: raw pork and poultry meat, raw sheep milk cured cheese, and ready to eat lettuce salad. Four parameters were evaluated to reduce the time and cost for final results: the initial sample size (25 and 50 g), the dilution of the sample (1:3; 1:5 and 1:10 dilutions in Half Fraser broth), the incubation times (6, 10 and 24h) and the bacterial DNA extraction (simple boiling of the culture after washing the bacterial pellet, the use of the Chelex resin, and a commercial silica column. The results obtained demonstrate that a combination of an incubation in Half-Fraser for 24h of a 1:10 diluted-25 g-sample coupled to a DNA extraction using a commercial silica column and a real-time PCR assay detected down to 2-4 L. monocytogenes CFU per sample in less than 27 h in different types of food products. This RTi-PCR-based method is fully compatible with the ISO standard, providing results more rapidly and cost-effectively. The results were confirmed in a large number of naturally contaminated food samples with at least the same analytical performance as the reference method.