Ureteroscopic laser lithotripsy requires irrigation for adequate visualization and temperature control during treatment of ureteral stones. However, there are little data on how different irrigation and laser settings affect the ureteral wall and surrounding tissues. This effect has become an important consideration with the advent of high-powered lasers. We therefore evaluated the effect of laser settings and irrigation flow on ureteral temperature in an in vitro setting. To mimic ureteroscopic laser lithotripsy, we simulated clinically relevant irrigation flow rates and fired a Holmium:Yttrium-aluminum-garnet (Ho:YAG) laser while monitoring "intraureteral" temperature. The probe tip of a thermometer was placed 1 mm from the tip of a 200 μm laser fiber, which was fired for 60 seconds at 0.2 J/50 Hz, 0.6 J/6 Hz, 0.8 J/8 Hz, 1 J/10 Hz, and 1 J/20 Hz within a tubing system that allowed for specified room temperature flow rates (100, 50, and 0 mL/minute). We recorded temperatures every 5 seconds. The maximum temperature was noted, and each laser/flow trial was duplicated. Averaged maximum temperatures were compared using analysis of variance across irrigation settings. At 100 cc/minute, only the 1 J/20 Hz laser setting produced a significantly higher maximum temperature (p < 0.01), although this finding was not clinically significant at a maximum of 30.7°C. At a lower irrigation rate of 50 cc/minute, the 1 J/20 Hz setting was again the only significantly higher maximum temperature (p < 0.05), although this temperature crossed the toxic threshold at a maximum of 43.4°C. With no flow, all maximum temperatures reached over 43°C, with 0.8 J/8 Hz, 1 J/10 Hz, and 1 J/20 Hz each statistically higher than the lower-energy settings (p < 0.05). The maximum temperature at 1 J/20 Hz with no irrigation was over 100°C. Despite increasing laser settings, adequate irrigation can maintain relatively stable temperatures within an in vitro ureteral system. As irrigation rates decrease, even lower power laser settings produce a clinically significant increase in maximum temperature, potentially causing ureteral tissue injury.