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Progesterone (PG) diminishes free radical damage and thus can afford protection against oxidative stress affecting the retina. The therapeutic use of PG is limited because it is a highly hydrophobic steroid hormone with very low solubility in water. This is the main drawback for the therapeutic application of PG at ocular level. The aims of this study were: (i) to analyze if PG causes ocular irritation (ii) to validate a HPLC method to determine PG in ex vivo studies and (iii) to evaluate PG permeation through cornea and sclera. A high performance liquid chromatographic method was developed and validated to detect PG incorporated to β-cyclodextrin using a Waters Sunfire C18 (150 × 4.6 mm) reverse-phase column packed with 5 μm silica particles using a mobile phase consisted of a mixture of acetonitrile (ACN) and pure water 80:20 (v/v), pH 7.4. The limit of detection and the limit of quantification for 50 μL injection of PG were found to be 0.42 and 1.26 μg/mL, respectively. The calibration curve showed excellent linearity over the concentration range (0.5 μg/mL to 100 μg/mL). As proof of concept, ex-vivo experiments to investigate PG permeation through cornea and sclera with vertical diffusion cells were carried out to quantify PG diffusion. Ex vivo experiments demonstrate its applicability to investigate permeation levels of PG from 6.57 ± 0.37 μg/cm 2 at cornea and 8.13 ± 0.85 μg/cm 2 sclera. In addition, at the end of diffusion studies the amount of PG retained in each tissue was also quantified, and it was 40.87 ± 9.84 μg/cm 2 (mean ± SD; n = 6) in cornea and 56.11 ± 16.67 μg/cm 2 (mean ± SD; n = 6) in sclera., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. Only the authors played a role in the design of the study, in the collection, analyses, interpretation of data as well as in the writing of the manuscript, (Copyright © 2020 Elsevier B.V. All rights reserved.)