Purpose <p>This post hoc analysis aimed to explore the effect of perfluorohexyloctane on dry eye disease (DED) associated with meibomian gland dysfunction (MGD) by baseline disease severity.</p> Methods <p>Data from a randomized phase 3 trial (NCT05515471) were divided based on the baseline severity of DED signs (total corneal fluorescein staining [tCFS] score: &lt; 6 or ≥ 6; tear film breakup time: &lt; 3 or ≥ 3; MGD score: &lt; 7 or ≥ 7) or symptoms (eye dryness scores [EDS]: &lt; 70 or ≥ 70; ocular surface disease index score: &lt; 60 or ≥ 60). The main outcomes of interest included response rates at day 57 of tCFS score, EDS, and tCFS and EDS, safety, and tolerability.</p> Results <p>Participants treated with perfluorohexyloctane had greater odds of achieving EDS (odds ratio [OR]: 2.25; 95% confidence interval [CI]: 1.34, 3.80), tCFS (OR: 2.21; 95% CI: 1.38, 3.55), and tCFS and EDS (OR: 2.56; 95% CI: 1.62, 4.04) responses than those treated with 0.6% sodium chloride in the overall population. No significant treatment interaction effect was observed by any DED signs or symptoms except for a significant effect of baseline tCFS score on EDS response, with a relatively large effect in participants with tCFS ≥ 6 versus tCFS &lt; 6 (propensity score-weighted OR: 4.35 versus 1.26; P_interaction = 0.0317). The safety and tolerability profiles of perfluorohexyloctane stratified by baseline disease severity were generally consistent.</p> Conclusion <p>Perfluorohexyloctane was well tolerated and improved the signs and symptoms of DED associated with MGD in patients with different baseline disease severity compared to saline control.</p>