<p>Narrow therapeutic index drugs (NTIDs) require precise monitoring to ensure efficacy and prevent toxicity, yet their analysis in complex biological matrices remains challenging. This study presents a novel sonic-spray ionization mass spectrometry (SSI-MS) method for the rapid and sensitive analysis of NTIDs, using tacrolimus as a model compound. The custom-built SSI source operates without high voltage or heating, relying solely on a coaxial nitrogen flow for gentle and efficient ionization. Under optimized conditions, the method achieved a low limit of quantitation (5 ng/mL) and excellent linearity (R² = 0.999) across the clinically relevant therapeutic window (5–200 ng/mL). Compared to electrospray ionization, SSI provided a cleaner background and a 1.88-fold improvement in the signal-to-noise ratio. The accuracy of this method was validated in mice serum, with recoveries ranging from 99.8% to 106.8% and relative standard deviations below 8.4%. Furthermore, the platform successfully detected tacrolimus directly in a saline matrix. These results demonstrate that SSI-MS is a powerful, high-performance tool for the rapid and reliable quantification of NTIDs, offering significant potential for advancing therapeutic drug monitoring in clinical practice.</p> Graphical abstract