Featured Application Malignant and benign thyroid nodules classification. A rapid classification method was developed for the malignant and benign thyroid nodules with ultrasound guided-fine needle aspiration biopsy (FNAB) samples. With probe electrospray ionization mass spectrometry, the mass-scan data of FNAB samples were used as datasets for machine learning. The patients were marked as malignant (98 patients), benign (110 patients) or undetermined (42 patients) by experienced doctors in terms of ultrasound, the B-Raf (BRAF) gene, and cytopathology inspections. Pairwise coupling was performed on 163 ions to generate 3630 ion ratios as new features for classifier training. With the new features, the performance of the multilayer perception (MLP) classifier is much better than that with the 163 ions as features directly. After training, the accuracy of the MLP classifier is as high as 92.0%. The accuracy of the single-blind test is 82.4%, which proved the good generalization ability of the MLP classifier. The overall concordance is 73.0% between prediction and six-month follow-up for patients in the undetermined group. Especially, the classifier showed high accuracy for the undetermined patients with suspicious for papillary carcinoma diagnosis (90.9%). In summary, the machine learning method based on FNAB samples has potential for real clinical applications.