Abstract: Hydrocortisone is an important biomarker in clinical medicine and pharmacology， but the traceability of hydrocortisone hasn’t been settled yet. Identification and quantification of impurities in hydrocortisone as well as purity of hydrocortisone were studied in this work. Structure analysis and identification of related structure impurities were carried out by HPLC-IT-TOF. Related structure impurities were quantified by HPLC-MS/MS. The methods of Karl Fischer Coulometric, headspace GC and ICP-MS were used for quantification of other impurities. Purity of hydrocortisone is concluded to be 99.18% by mass balance method with an expended uncertainty of U=0.08% (k=2). The result was further confirmed by quantitative NMR spectroscopy method. The results of two methods are in good agreement, which indicate that qualitative and quantitative analysis method established in this work is accurate and reliable. Moreover, this method has instructive significance to the qualitative and quantitative analysis of pure steroids and other pure organic substances.