Currently commercial fixed-concomitant three agents have multiple problems such as multiple dosing administration, poor efficacy and side effects. Once-daily fixed-combination timolol-netarsudil-latanoprost ophthalmic solution (FC-TNL) has the ability to treat glaucoma by lowering the intraocular pressure (IOP) with great efficacy and improving patient compliance. However, the commercialized netarsudil dimesylate precipitated when the pH of the solution was above 5.4, or when maleic acid, the salt of commercial timolol maleate, was mixed with netarsudil dimesylate. Consequently, the homologous salt engineering strategy was used to make netarsudil dimesylate soluble in pH 4.8-5.2 solution by synthesizing timolol mesylate. Next, the morphology of timolol mesylate was observed by scanning electron microscopy, differential scanning calorimetry, thermogravimetric analysis, and powder X-ray diffraction. The prepared FC-TNL showed good stability during refrigeration storage. Additionally, FC-TNL exerted no influence on the intraocular penetration of each active compounds in the pharmacokinetic study. Importantly, once - daily FC-TNL exerted potent IOP-lowering effect and protective effect on retinal ganglion cells. The FC-TNL was stable, safe and effective, being a promising glaucoma therapeutic.(c) 2022 Published by Elsevier B.V. on behalf of Shenyang Pharmaceutical University. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )