Abstract: Objective To develop a qualitative and quantitative analytical method for six grayanane diterpenoid toxins using UPLC-MS/MS and apply it to honey samples from poisoning incidents. Clinical symptoms of the patients were analyzed in combination with field epidemiological findings. Methods Honey samples were extracted and purified using the QuEChERS method, followed by qualitative and quantitative analysis with UPLC-MS/MS. Patient clinical manifestations were assessed based on field epidemiological investigations and laboratory reports. Results The validated method exhibited excellent linearity (R2 ≥ 0.997) over the range of 1–400 ng/mL. The limits of detection (LOD) and quantification (LOQ) were 0.13–0.30 ng/mL and 0.44–0.99 ng/mL, respectively. Spiked recoveries ranged from 90.6% to 107.6% with relative standard deviations (RSD) ≤ 6.6%. Grayanotoxin III was detected in honey samples at concentrations of 0.606–43.976 mg/kg. Patients presented symptoms consistent with grayanotoxin III poisoning, including dizziness, peripheral paresthesia, blurred vision, palpitations, and sinus bradycardia. Conclusion The established method is rapid, sensitive, and reliable for grayanotoxin detection. This poisoning incident was confirmed to be caused by grayanotoxin III contamination in honey.