摘要TEM and SHV are among the most prevalent β-lactamases contributing to β-lactam antibiotic resistance in clinical settings,leading to treatment challenges and increased mortality rates.Except for penicillin and early cephalosporins,TEM and SHV variants have evolved with the ability to hydrolyze the second-and third-generation cephalosporins,monobactams,and even β-lactamase inhibitors.Accurate detection of β-lactamases is of paramount importance for optimizing antibiotic use and combating antimicrobial resistance(AMR).While genetic detec-tion methods,such as polymerase chain reaction(PCR),are widely employed,their positive results may lack phenotypic correlation due to the low or absent expression of blasHV and blaTEM in many strains[1].Therefore,a direct protein-level detection method such as targeted proteomics is more precise and clinically relevant.This study highlights the development of a rapid detection method using targeted proteomics with high-resolution accurate mass(HRAM)Orbitrap MS for the direct detection of TEM and SHV in Enterobacteriaceae strains,which offers greater clinical relevance compared to conventional ge-netic approaches.