Background Proparacaine hydrochloric ophthalmic solution (0.5%) is widely used as ophthalmic anesthetic for short conjunctival and corneal procedures. In connection to this, present research work was conducted to investigate ocular toxicity of its probable degradation products (DPs), which were generated after treating drug with stressors mentioned under International Conference on Harmonization (ICH) guideline Q1A (R2). Result The probable DPs were generated by placing drug under various stress conditions to generate similarity of the effect of stressor which drug supposed to face during its life cycle. The hydrolytic stress condition under acidic, basic and neutral environment formed two major DPs, while the drug was stable and did not generate any DP when subjected to oxidative, dry heat and photolytic stress. The separation of DPs was carried out using high performance liquid chromatographic system. The structural identification of DPs was carried out by subjecting degradation samples to liquid chromatography coupled with mass spectrometry. These DPs were screened using in-silico ocular toxicity prediction models Pub-SVM to predict their potential to cause irritation/corrosion to eye. Conclusions The DPs identified were DP 1 (2-(diethyl amino) ethanol) and DP 2 (3-amino-4-propoxybenzoic acid). The in-silico ocular toxicity study predicted that DP 1 has potential of eye corrosion (EC) and eye irritation (EI), whereas DP 2 has a potential EI. The drug was found safe and devoid of any EC and EI potential. © The Author(s) 2022. Published by Oxford University Press. All rights reserved.