We employed two commercially available femtosecond lasers, a Ti:sapphire and a ytterbium-based oscillator, to directly compare from a user’s practical point-of-view in one common experimental setup the efficiencies of transient laser-induced cell membrane permeabilization, i.e., of so-called optoporation. The experimental setup consisted of a modified multiphoton laser-scanning microscope employing high-NA focusing optics. An automatic cell irradiation procedure was realized with custom-made software that identified cell positions and controlled relevant hardware components. The Ti:sapphire and ytterbium-based oscillators generated broadband sub-15-fs pulses around 800 nm and 250-fs pulses at 1044 nm, respectively. A higher optoporation rate and posttreatment viability were observed for the shorter fs pulses, confirming the importance of multiphoton effects for efficient optoporation.