This method was validated in twenty patients with advanced HER2-positive breast cancer (dose range: 240–400 mg/day) The trough and peak plasma concentrations of pyrotinib ranged from 17.75–92.56 ng/mL and 51.17–232.94 ng/mL, respectively, which demonstrated significant pharmacokinetic heterogeneity. The gene discussed is ERBB2; the disease is breast carcinoma.