The aim of this novel in vivo study was to characterize the effect of short pulse-width versus long pulse-width Holmium-YAG laser lithotripter settings on retropulsion and fragmentation in a real-life setting. A prospective, crossover study was conducted at a tertiary teaching hospital in NSW, Australia. Patients who underwent flexible ureteroscopy with laser lithotripsy for renal calculi in 2018 were included. All patients underwent flexible ureteroscopy using a Flexor® 10.7/12 French ureteric access sheath (Cook Medical LLC, Bloomington, IN, USA) and lithotripsy with a 30W Holmium-YAG laser (Rocamed, Monaco). Thirty-two renal calculi were subjected to 1 min of laser treatment using both short and long pulse-width settings. Using 5-point, operator-assessed Likert scales, the level of retropulsion and fragmentation efficacy were assessed. There was significantly less retropulsion and improved stone fragmentation (p<0.001) using the long pulse-width compared to the short pulse-width setting. Regardless of stone size, in vivo renal calculi lithotripsy with a long pulse-width significantly improves the efficacy of lithotripter treatment.