The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae represents a major public health concern, primarily driven by its ability to evade a wide range of antibiotics. Despite extensive genomic studies, proteomic insights into antibiotic resistance mechanisms remain scarce. Here, we employed a data-independent acquisition (DIA)-based quantitative proteomics approach to investigate proteomic differences between 87 MDR and 20 antibiotic-sensitive K. pneumoniae clinical isolates. A total of 3,380 proteins were identified, with 896 showing significant differential expression. MDR isolates exhibited increased expression of efflux pumps, beta-lactamases, and transcriptional regulators, while proteins associated with glycerolipid metabolism and transport were enriched in sensitive strains. To validate our findings, an independent cohort of 10 MDR and 11 sensitive isolates was analyzed. Key biomarkers identified in the discovery cohort, including pyruvate decarboxylase and aldehyde dehydrogenase, were validated with high discriminatory power (AUC > 0.85) in the validation cohort. These findings provide novel insights into the molecular mechanisms of antibiotic resistance and identify promising biomarkers for diagnosing MDR K. pneumoniae, offering potential avenues for therapeutic intervention.