Background <p>Non-tuberculous mycobacteria (NTM) are of increasing clinical importance, yet their identification remains challenging. While matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers a rapid, high-throughput solution, its diagnostic accuracy for NTM has not been fully established.</p> Methods <p>A comprehensive systematic review and meta-analysis published between 2011 and May 2024 were conducted. Studies evaluating the diagnostic accuracy of MALDI-TOF MS for NTM species identification against reference standards were included. Pooled identification rates were calculated via a random effects model. Subgroup analyses were used to assess the influence of variables such as growth rate, culture media, and instrument platform. Quality assessment was performed via the Joanna Briggs Institute checklist.</p> Results <p>Across 55 studies, MALDI-TOF MS achieved a pooled identification accuracy of 88% (95% CI: 84–91%) for clinical NTM isolates. Subgroup analyses revealed greater accuracy for rapidly growing mycobacteria (93%) than for slowly growing mycobacteria (89%). Compared with liquid (85%) or co-culture (84%) systems, cultures on solid media yielded superior results (91%). The Bruker MALDI Biotyper and bioMérieux VITEK MS platforms showed comparable performance, although species-level accuracy varied. Notably, species such as <i>M. fortuitum</i>, <i>M. smegmatis</i>, and <i>M. marinum</i> were identified with high precision (&gt; 95%), whereas species such as <i>M. colombiense</i> and <i>M. parascrofulaceum</i> exhibited poor discrimination.</p> Conclusion <p>MALDI-TOF MS is highly accurate in identifying clinically relevant NTM, particularly rapidly growing species. Despite improvements in databases and sample preparation, distinguishing closely related or rare species remains challenging, underscoring the need for further optimization and standardization.</p>