Milk thistle (Silybum marianum (L.) Gaertn.), a valuable medicinal plant with economic importance, serves as a source of both therapeutic compounds and vegetable oil for human use. Genome editing techniques have been widely applied to improve key agronomic traits in various species. Therefore, this study aims to develop an efficient method for seed sterilization and protoplast isolation in milk thistle. For seed sterilization, seed treated with hydrogen peroxide for either 4 hours or 7 hours with ethanol and detergent showed more sufficient to sterilize seed of milk thistle for in vitro growth than ethanol, sodium hypochlorite, and chlorine gas. For protoplast isolation in milk thistle, high-yielding, viable protoplasts were successfully isolated from the leaves of 21-day-old plants using a 2 hours enzymatic treatment containing 2% Viscozyme^® L, 1% Celluclast^® 1.5 L, and 1% Pectinex^® Ultra SP-L. Additionally, transient expression of green fluorescent protein was observed following polyethylene glycol-mediated transfection. Therefore, the seed sterilization and protoplast isolation methods developed in this study can facilitate in vitro culture, gene function analysis, and genome editing aimed at improving agronomic traits in milk thistle.