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, , and fluence thresholds, , 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 , , , and . 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.