Purpose <p>This review critically evaluates the performance of direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid pathogen identification in life-threatening infections. It aims to assess its application directly from positive blood cultures and sterile body fluids, examine the factors influencing identification success rates, and identify the key challenges limiting its broader clinical implementation.</p> Methods <p>A review of the literature was conducted, focusing on studies utilizing commercial kits or optimized laboratory protocols for direct testing from clinical specimens, including positive blood cultures, cerebrospinal fluid, urine, and enrichment cultures from synovial fluid and ascites.</p> Results <p>Direct MALDI-TOF MS can deliver reliable pathogen identification within 30–60 minutes, significantly shortening diagnostic turnaround time. However, identification success is not uniform; a consistent performance gradient is observed, with Gram-negative bacteria identified more successfully than Gram-positive bacteria and fungi. This variability reflects differences in microbial cell structure and protein extraction efficiency. Major constraints include limited sensitivity in paucibacterial samples, spectral interference from host matrices, difficulties in diagnosing polymicrobial infections, and gaps in reference databases for emerging pathogens.</p> Conclusion <p>Direct MALDI-TOF MS is a transformative tool for rapid infection diagnosis. To fully realize its potential in routine practice, future advancements must integrate automated sample processing, enhance sensitivity via techniques like MALDI-2, and employ artificial intelligence for spectral analysis and resistance prediction. Overcoming these barriers will establish this technology as a cornerstone of rapid diagnostic pathways, improving patient outcomes and supporting antimicrobial stewardship.</p>