Risdiplam, the first oral therapy approved for spinal muscular atrophy and made globally available in 2021, necessitates a highly sensitive and straightforward assay for therapeutic drug monitoring. This is crucial to manage potential toxicities linked to drug concentrations and supervise dosing regimens. A cutting-edge ultra-high performance liquid chromatography-tandem mass spectrometry bioassay for risdiplam in human serum has been developed. In this method, analytes were separated on a Phenomenex Kinetex XB C18 column using a 6.5-min gradient elution after a single-step protein precipitation. MS detection was conducted via electrospray ionization in positive mode with selected reaction monitoring. The validated range for risdiplam was determined to be 1.95-125.00 ng/mL. The precision and accuracy of intra- and inter-batch analyses were within ±15%. The novel method met all other established criteria. This assay holds promise for monitoring drug concentrations and guiding clinical decisions in patients with spinal muscular atrophy.