Drug-induced liver injury (DILI) pertains to liver damage resulting from xenobiotics like antibiotics. The current study aimed to formulate and assess fast-dissolving sublingual niosomal film (FSNF) containing rutin (RT) and evaluate its hepatoprotective effect against ceftriaxone-induced hepatotoxicity in rats. FSNF was utilized to enhance the poor solubility, absorption, and oral bioavailability of RT. RT-loaded niosomes were prepared using both Span 40 and 60 at various cholesterol concentrations. The niosomes were characterized for entrapment efficiency %, particle size, zeta potential, polydispersity index, and in vitro release. An optimal formulation of RT-loaded niosomes, containing 29.3 % cholesterol and 250 μmoles total lipids with Span 60 as a surfactant, was selected following an I-optimal experimental design. This formulation was subsequently integrated into a selected polymeric film made with hydroxypropyl methylcellulose K15M and propylene glycol as a plasticizer. An in vivo study conducted in albino male rats aimed to explore the pharmacokinetic parameters and the hepatoprotective activity of RT-FSNF versus oral pure RT suspension. RT-FSNF notably enhanced the absorption and oral bioavailability of RT, as evidenced by the elevated mean Cmax and AUC0-t observed after sublingual administration of RT-FSNF. Moreover, the mean Tmax, t1/2, and MRT were significantly increased suggesting the sustained release impact of RT-FSNF compared to oral pure RT suspension. Additionally, the in vivo study demonstrated that RT-FSNF exhibited enhanced hepatoprotective effects, evidenced by the reduced serum liver enzymes and its capability to restore liver antioxidant activity. These findings suggest that RT-FSNF may serve as an effective delivery system to enhance oral bioavailability, improve hepatoprotective properties, and extend the therapeutic effects of RT.