A rapid, precise, and sensitive liquid chromatography/mass spectrometry (LC/MS) method to quantify the caspofungin concentration in human aqueous humor was developed and validated. Sample preparation involved simple dilution of aqueous humor samples with acetonitrile. Azithromycin was the internal standard. Good linearity over 10 to 5,000 ng/ml was observed. The lower limit of quantification was 10 ng/ml. The intra- and interday accuracies (percent bias) were within 11%, while the intra- and interday precisions were within 6%.