The work aims to establish a high performance liquid chromatography (HPLC) method to detect different antioxidants and extractable sulfur in medicinal rubber plugs simultaneously. Dichloromethane was used as extraction solvent. The target compounds in medicinal rubber plugs were extracted via microwave method. Content of target was tested by the HPLC via gradient elution. Waters Symmetry RP 18 was used as chromatographic column. Acetonitrile, methanol and distilled water were used as mobile phases. The detection wave was set to 277 nm. The target compounds can be separated by HPLC efficiently. The separated products possessed excellent resolution. There was a favourable linearity in the range of 0.2-20 μg/mL with the correlation coefficient r ≥ 0.999 3. The detection limit of this method was 0.03-0.09 μg/mL, the rate of recovery was 86.73%-108.91% and the relative standard deviation (RSD) was 0.47%-5.68%. Medicinal rubber plugs with different batches from different manufacturers were detected by this method. As a results, antioxidant (BHT) and its degradation products (BHT-CHO, BHT-Q), antioxidants (1076 and 1010) and their degradation product (1310), as well as extractable sulfur were detected.The HPLC method established in this work is accurate, convenient and highly sensitive. It can determine the contents of antioxidants and their degradation products and extractable sulfur in common medicinal rubber plugs rapidly and efficiency.