Background Prosaikogenin F (PSF) has stronger anti-cancer bioactivity than saikosaponin A (SSA), however, it was hardly isolated due to its trace amount in the raw material of Radix Bupleuri (RB). In addition, the active chemical constituent was unstable under acidic conditions owing to a 13,28-epoxy-ether moiety at the D ring of its aglycone. Objectives This study was to develop an appropriate method for obtaining acid-sensitive PSF from SSA abundant in RB. Materials and Methods Enzymatic hydrolysis was employed and snailase was selected due to its good hydrolysis performance under nearly neutral circumstances. Hydrolysis conditions were then optimized by one-factor-at-a-time experimentation before response surface methodology (RSM) by Box-Behnken Design (BBD). Results The reaction system was constructed in Na2HPO4-NaH2PO4 buffer (pH 6.0) containing snailase/SSA (44:1) at 39°C, then the hydrolysis lasted for 12 h. Therefore, the highest conversion ratio of SSA was achieved at 100.0%. Conclusion The newly proposed method is eco-friendly for obtaining acid-sensitive PSF, which lays a solid foundation for its development to be an anti-cancer new drug.