Biotransformation of colchicine into its pharmacologically active regiospecific derivative 3‐demethylated colchicine (3‐DMC) mediated by microbial monooxygenases is an economic and promising strategy for the production of this inexpensive and potent anti‐cancer drug. Response surface methodology (RSM), with central composite design (CCD) model, was employed with three extraction variables (temperature, pH, and process time) for optimization; which most significantly affected recovery of 3‐DMC. The validity of the model was ascertained by running the predicted values in an individual run; and the optimum parameters, temperature (50°C), pH (10), and process time (120 min) resulted in a maximum recovery of 3‐DMC 4128 mg/L when chloroform (1% v/v) was used as an extraction solvent. RSM in our experiments showed to be a good tool for the extraction of 3‐DMC from the fermentation medium.