A method based on an offline coupling of high-performance liquid chromatography/electrospray ionization multi-stage mass spectrometry (HPLC-ESI-MSn) and high-speed counter-current chromatography (HSCCC), was established for targeted separation and purification of skin-irritative and structurally similar ingenol-type diterpenoids in Euphorbia kansui. The crude dichloromethane extract of Euphorbia kansui after an initial clean-up step on silica gel and targeted fractionation according to HPLC-ESI-MSn, was separated by HSCCC. As a result, 3-O-(2E,4Z-decadienoyl)-20-O-acetylingenol and 3-O-(2E,4E-decadienoyl)-20-O-acetylingenol as well as a pair of geometrical isomers, 5-O-(2E,4Z-decadienoyl)-20-O-acetylingenol and 5-O-(2E,4E-decadienoyl)-20-O-acetylingenol, were successfully separated and purified. The established method is rapid and efficient without irritation to operators, providing an extensible and feasible way for the separation and purification of other toxic and skin-irritative compounds.