Abstract: Theophylline is a widely used medication for treating asthma and chronic obstructive pulmonary disease (COPD). It’s essential to accurately monitor the blood concentration of theophylline, due to the significant inter-individual variability and its narrow therapeutic window. Currently, immunoassays, high-performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are commonly used as routine methods for measuring serum theophylline in clinical laboratories. However, there is significant difference among the measurement results of these methods. The reference measurement procedure for serum theophylline in the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database is the isotope dilution gas chromatography-mass spectrometry (ID-GC/MS) method. The primary reference material was developed by the UK Laboratory of the Government Chemist (LGC) and China National Institute of Metrology (NIM). No secondary reference material with serum matrix was reported. To ensure the reliability and harmonization of measurement results, it is important to establish a comprehensive traceability chain for the standardization of serum theophylline.