We wished to determine whether Resonant Raman Spectroscopy (RRS) could be used to measure Amphotericin B (AmB) at therapeutic and subtherapeutic concentrations in a model system mimicking the anterior chamber of the eye. The goal was to develop a technique for non-invasive measurement of AmB levels in the aqueous humor (AH) of the eye. A krypton-ion laser source (406.7 nm) was used for excitation and Resonant Raman Spectra were captured with a confocal system in an anterior chamber (AC) model. These spectra were used to develop a correlation curve for prediction of AmB levels. Subsequently, one rabbit was evaluated with this system after 5 days of intravenous AmB administration (1 mg/kg/day) and AmB concentrations measured by RRS were compared to those measured by high-performance liquid chromatography (HPLC). AmB exhibited a unique spectral peak at 1557 cm(-1). Integrated area of this peak linearly correlated with AmB concentration in our model AC. When integrated peak area from multiple in vivo measurements in one animal at steady-state was plotted on this correlation curve, we were able to predict AmB levels. These closely approximated those measured by HPLC. These measurements were not significantly affected by photobleaching or depth profile at acquisition. RRS at 406.7 nm is a method that may be useful for non-invasive monitoring of intraocular AmB levels. This instrument can help physicians decide when repeat, invasive delivery of this drug is warranted based on measurement of actual drug levels in the AH. Also, there is the potential to measure the ocular concentrations of other pharmaceutical agents with similar instruments.