Background and Objectives: Femtosecond laser trabeculotomy (FLT) creates aqueous humor outflow channels through the trabecular meshwork (TM) and is an emerging noninvasive treatment for open-angle glaucoma. The purpose of this study is to investigate the effect of pulse energy on outflow channel creation during FLT., Materials and Methods: An FLT laser (ViaLase Inc.) was used to create outflow channels through the TM (500 μm wide by 200 μm high) in human cadaver eyes using pulse energies of 10, 15, and 20 μJ. Following treatment, tissues were fixed in 4% paraformaldehyde. The channels were imaged using optical coherence tomography (OCT) and assessed as full thickness, partial thickness, or not observable., Results: Pulse energies of 15 and 20 μJ had a 100% success rate in creating full-thickness FLT channels as imaged by OCT. A pulse energy of 10 μJ resulted in no channels (n = 6), a partial-thickness channel (n = 2), and a full-thickness FLT channel (n = 2). There was a statistically significant difference in cutting widths between the 10 and 15 μJ groups (p < 0.0001), as well as between the 10 and 20 μJ groups (p < 0.0001). However, there was no statistically significant difference between the 15 and 20 μJ groups (p = 0.416)., Conclusions: Fifteen microjoules is an adequate pulse energy to reliably create aqueous humor outflow channels during FLT in human cadaver eyes. OCT is a valuable tool when evaluating FLT., (© 2024 Wiley Periodicals LLC.)