The study investigated the effects of cutting the pre-Descemet's layer (PDL) and Descemet's membrane (DM) on aqueous outflow from the anterior chamber of the eye using the anterior segment perfusion model (ASPM) in human donor eyes. In the experimental eyes, the PDL and DM were cut along the circumference of a circle with a diameter of 8 mm at an angle of 180° in one half of the eye, while the other half remained uncut. Experimental eyes showed a decrease in outflow velocity, indicating increased outflow resistance. Expression of fibronectin, myocilin, and alpha-smooth muscle actin was increased in the trabecular meshwork (TM) of cut eyes compared to uncut and control samples. In the outflow perfusates of the experimental eyes, the level of active MMP-9 was significantly higher on day 4 and decreased on day 9 compared to controls. Myocilin was detected in the perfusate of experimental eyes up to day 9. Disruption of the PDL and DM resulted in reduced outflow caused by changes in biomechanical properties and molecular changes in the TM, which may explain the higher incidence of increased pressure in penetrating keratoplasty versus deep anterior lamellar keratoplasty.