Background/Objectives: Carbapenem-resistant Enterobacterales, largely due to carbapenemase production, are significant public health threats, which compromise treatment with key β-lactam antibiotics. Early detection is essential for guiding therapy and controlling spread. This study describes the design, optimisation and validation of a multiplex real-time PCR for the screening of the most frequent carbapenemases in our area. Methods: Primers and probes targeted at genes encoding carbapenemases bla_KPC, bla_IMP, bla_VIM, bla_NDM and bla_OXA-48-group were designed and adapted for the development, and in silico and experimental validation of a single-tube real-time PCR. Results: A good linear correlation between the fluorescence values in the real-time PCR and the log₁₀ of bacterial concentration of each carbapenemase-containing bacterial suspension was observed (R² > 0.98). The limit of detection was 2–15, 16–256, 42–184, 4–42, 42–226 CFU/reaction of VIM-, IMP-, NDM-, KPC- and OXA-48-carbapenemase-containing bacteria, respectively. Intra-assay coefficient of variation for the mean Ct values ranged from 0.99% for OXA-48 to 3.34% for KPC. Inter-assay variability remained below 7%. Real-time PCR tested on bacterial isolates yielded 100% sensitivity and specificity. Analysis of rectal swabs using extracted DNA and a DNA extraction-free protocol showed good concordance with culture-based phenotypic methods. Additionally, the molecular method could detect all targets, except for one sample where only the DNA extraction-free protocol detected NDM. Conclusions: The assay offers a rapid, sensitive and specific method for the screening of major carbapenemase genes, providing an effective tool for surveillance and infection control in clinical settings. The DNA extraction-free protocol converts this method into a good alternative for screening in 24/7 clinical laboratories. Further multiplexing to target other resistance genes, on demand, could add potential benefits to this laboratory-developed method.