A method for optimizing an inspection recipe of a defect inspection tool is described. A substrate having thereon intentional defects and locating patterns beside the intentional defects is provided. The defect inspection tool is used to detect the intentional defects with an inspection recipe and obtain the distribution of undetected or partially detected intentional defects. The locating patterns are utilized to locate the undetected or partially detected intentional defects and thereby determine the type(s) of the undetected or partially detected intentional defects. The inspection recipe is modified according to the type(s) of the undetected or partially detected intentional defects in a manner such that there is a minimal number of undetected or partially detected intentional defects under the inspection of the defect inspection tool.