We explored the potential of the picosecond Nd:YLF laser to perform gonioscopic ab interno sclerostomy in human cadaver eyes. Full-thickness sclerostomies were created in 12 such eyes and confirmed by external scleral and histopathologic examination. Optimum parameters for successful completion of a sclerostomy were: a rectangular pattern of 0.3 mm by 0.3 mm, a repetition rate of 1000 pulses per second, an energy-per-pulse of 400 mJ, and a spot separation between 10 and 50 microns, with no focal offset. The Nd:YLF laser reliably created successful gonioscopic sclerostomies with minimal adjacent thermal damage in human cadaver eyes.