Current information about the pharmacokinetics of an ocular drug can only be achieved by invasive sampling. However, confocal Raman spectroscopy bears the potential to quantify drug concentrations non-invasively. In this project, we evaluated the detection and quantification of ocular ketorolac tromethamine levels with confocal Raman spectroscopy after topical administration. Confocal Raman spectroscopy and high-performance liquid chromatography (HPLC) were compared in terms of sensitivity of detection. Enucleated pig eyes were treated with different concentrations of ketorolac. Hereafter, ketorolac concentrations in the aqueous humor of pig eyes were analyzed by confocal Raman spectroscopy and HPLC. Subsequently, twelve rabbits were treated with Acular™ for four weeks. At several time points, ketorolac concentrations in aqueous humor of the rabbits were measured by confocal Raman spectroscopy followed by drawing an aqueous humor sample for HPLC analysis. In ketorolac treated pig eyes, both ex vivo Raman spectroscopy as well as HPLC were able to detect ketorolac in a broad concentration range. However, in vivo confocal Raman spectroscopy in rabbits was unable to detect ketorolac in contrast to HPLC. To conclude, confocal Raman spectroscopy has the capacity to detect ketorolac tromethamine in vitro, but currently lacks sensitivity for in vivo detection.