Paper spray high-resolution accurate mass spectrometry is a fast and versatile analysis method. This ambient ionization technique enables the quantitation of xenobiotics in complex biological matrices without chromatography or conventional sample extraction. The simplicity, rapidity, and affordability of the paper spray mass spectrometry (PS-MS) method make the technique especially attractive for clinical investigations where fast and affordable sample analysis is crucial. A new PS-MS method for the quantitation of voriconazole in equine tears was developed and validated. For a concentration range of 10 to 1000 ng/mL, good linearity (R2 > 0.99), inter- and intra-run precision (coefficient of variation (CV) max. 11.9%), accuracy (bias of the nominal concentration ± 13.9%), and selectivity (signal areas of the double blanks represent 0.13 ± 0.05% of the lower limit of quantitation (LLOQ) signal in equine tears) were observed. The quantitation of voriconazole was based on three product ions and calculated relative to the isotope-labeled internal standard, voriconazole-d3, which had a final concentration of 250 ng/mL in the standards and samples. The matrix effect of the method showed an ionization suppression by reduction of the voriconazole response to 63.6%, 70.2%, and 81.9% for 30 ng/mL, 450 ng/mL, and 900 ng/mL in equine tears compared with voriconazole in solvent (methanol:water, 50:50, v:v). The method was used to analyze 126 study samples collected for a pharmacokinetic study investigating a novel approach for treatment of fungal keratitis in horses. Therefore, the integrity of the sample dilution (n = 6,CV 6.90%, and bias of nominal concentration + 8.40%) and the carryover effect (increase from 0.33 ± 0.21% to 1.33 ± 0.89% of the signal of the LLOQ) was further investigated. To our knowledge, this method is the first application of PS-MS for quantitation of drug concentrations in tears from any species.