OBJECTIVE： To establish a method for the concentration determination of isavuconazole in rabbit cornea. METHODS： HPLC was performed on column of Waters X-Bridge C18 with mobile phase of 0.2％ glacial acetic acid-acetonitrile （49 ∶ 51， V/V） at flow rate of 0.7 mL/min， column temperature was 40 ℃， detection wavelength was 284 nm， and injection volume was 10 μL. Fifteen Japanese white rabbits with big ears were selected， 50 μL of 0.2％ isavuconazole suspension was dropped into eyes， then every 3 rabbits were executed after 5， 15， 30， 60， 90 min of administration and cornea samples were taken respectively. The determination of isavuconazole were determined after treating. RESULTS： The isavuconazole in leaching solution showed good linear range in 0.031 2-2.24 μg/mL （r2＝0.999 9）； oethod recovery rate was 91.7％-105.6％ （RSD≤6.88％， n＝5）； extraction recovery rate was 100.50％-106.10％ （RSD≤2.16％， n＝3）； and RSDs of inter-day （n＝3） and intra-day （n＝3） precision and stability （n＝5） tests were lower than 10％. CONCLUSIONS： The method shows strong specificity， high accuracy， good reproducibility， and can rapidly and accurately determine the concentration of isavuconazole in rabbit cornea.