Abstract： Background Elevation of intraocular pressure is usually associated with primary open angle glaucoma and caused by increased outflow resistance. A two-color fluorescent tracer technique was developed to investigate the hydrodynamics of aqueous humor outflow with changing intraocular pressure within the same eye, to better understand the relationship between outflow facility and effective filtration area. Methods Eighteen enucleated bovine eyes were first perfused at 30 mmHg with Dulbecco's phosphate-buffered saline containing 5.5 mmol/L D-glucose. After a stable baseline facility, red fluorescent microspheres (0.5 μm, 0.002% v/v) were exchanged and perfused. Eyes in the one-color control group (n=6) were immediately perfused with fixative. In the experimental group (n=6), eyes were perfused with green tracer after intraocular pressure reduced to 7 mmHg, while in the two-color control group (n=6), eyes were perfused with green tracer with intraocular pressure remaining at 30 mmHg. All 12 eyes were then perfusion-fixed. Outflow facility was continuously recorded in all eyes. Confocal images were taken along the inner wall of the aqueous plexus and the percent of the effective filtration length (PEFL; length of inner wall exhibiting tracer labeling/total length of inner wall) was measured. The relationships between outflow facility and PEFL were analyzed statistically. Results No significant differences were found in baseline facilities (ulmin~(-1)·mmHg~(-1)) among the three groups (the experimental group: 0.93 0.12; the two-color control group: 0.90 0.19; the one-color control group: 0.98 0.13). In the experimental group, the outflow facility was significantly higher at 7 mmHg (4.29±1.01) than that at 30 mmHg (1.90±0.67, P <0.001), which corresponded to a significant increase in the PEFL at 7 mmHg (54.70±8.42) from that at 30 mmHg ((11.76±4.56)%, P<0.001). The PEFL labeled by red fluorescent microspheres in the experimental group ((11.76±4.56)%) showed no significant difference from that of the one-color control group ((13.39±2.19)%, P=0A73) or the two-color control group ((11.49±4.95)%, fM).93O). The PEFL labeled by green fluorescent microspheres in the experimental group ((54.70±8.42)%) was significantly higher than that of the two color control group ((37.34±8.17)%, A=0.010). A positive correlation was found between outflow facility and PEFL(r=3.897, R~2=0.804) in the experimental group. Conclusions Changes in aqueous humor outflow patterns before and after a change in intraocular pressure can be successfully distinguished within the same eye using our newly developed two-color tracer perfusion technique. The PEFL showed positive correlation with the outflow facility.