Ultrafast laser surgery of tissue requires precise knowledge of the tissue’s optical properties to control the extent of subsurface ablation. Here, we present a method to determine the scattering lengths, ℓs, and fluence thresholds, Fth, in multilayered and turbid tissue by finding the input energies required to initiate ablation at various depths in each tissue layer. We validated the method using tissue-mimicking phantoms and applied it to porcine vocal folds, which consist of an epithelial (ep) layer and a superficial lamina propia (SLP) layer. Across five vocal fold samples, we found ℓs,ep=51.0±3.9 μm, Fth,ep=1.78±0.08 J/cm2, ℓs,SLP=26.5±1.6 μm, and Fth,SLP=1.14±0.12 J/cm2. Our method can enable personalized determination of tissue optical properties in a clinical setting, leading to less patient-to-patient variability and more favorable outcomes in operations, such as femto-LASIK surgery.