This is the first study to evaluate the retention time of lubricating eye drops containing various concentrations of sodium hyaluronate using fluorophotometry in a symptomatic dry eye population. Information regarding eye drop retention may be useful for eye care practitioners to assist in the selection of more effective treatments for managing dry eye. This study aimed to use fluorophotometry to compare retention time on the ocular surface of three commercially available lubricating eye drops, each containing varying concentrations of sodium hyaluronic acid (HA), and their effects on tear film stability post-instillation in a population with symptoms of dry eye. Adults with symptoms of dry eye (Ocular Surface Disease Index score, >12) were enrolled in this prospective, double-masked comparison of eye drops containing 0.15% HA-hydroxypropyl guar (HPGuar), 0.2% HA, and 0.1% HA. Participants were randomized to the eye drop order and the study eye under evaluation. Each eye drop was admixed with a fluorescent tracer (70-kDa fluorescein isothiocyanate-dextran) at 10% wt/vol, and 10 μL volume was instilled for each evaluation. A fluorophotometer was used to measure the time for the tracer signal to return to baseline. Fluorescein tear breakup time was measured following fluorophotometry assessment. Retention time for 0.15% HA-HPGuar and 0.2% HA was significantly longer compared with 0.1% HA (p=0.02 and p=0.03). Fluorescein tear breakup time was significantly longer for the 0.15% HA-HPGuar eye drop compared with both the 0.1% HA eye drop (p=0.01) and 0.2% HA eye drop (p=0.003). Retention time on the ocular surface of the two eye drops containing higher concentrations of HA was longer than the eye drop with the lowest concentration of HA. The tear film was also more stable with the 0.15% HA-HPGuar eye drop compared with the eye drops containing HA alone, which may be attributable to the other components in the 0.15% HA-HPGuar eye drop.