Early antibiotic resistance determinants (ARDs) detection in humans or animals is crucial to counteract theirpropagation. The ARDs quantification is fundamental to understand the perturbation caused by disruptors,such as antibiotics, during therapies. Forty-three qPCRs on the most diffused ARDs and integrons amonghuman and animal Enterobacterales, and one on the 16S rDNA for bacteria quantification, were developed.The qPCRs, using hydrolysis probes, operated with a unique amplification condition and were tested analyticallyand diagnostically performing 435 reactions on five positive and negative controls for each qPCR. Diagnosticsensitivity and specificity were confirmed by PCR and genome sequencing of control isolates,demonstrating 100% performance for all qPCRs. An easy and rapid discrimination method for the epidemiologicallyrelevant blaCTX-Ms is provided. This large, noncommercial qPCRs inventory could serve for precisequantification of ARDs, but also as a rapid screening tool for surveillance purposes, providing the basis forfurther high-throughput developments.