A microscope-based system and a method for image-guided microscopic illumination are provided. The microscope-based system for image-guided microscopic illumination comprises a microscope, an illuminating assembly, an imaging assembly, a first processing module, and a second processing module. The microscope comprises a stage, and the stage is configured to be loaded with a sample. The imaging assembly comprises a camera. The processing modules are coupled to the microscope, the imaging assembly, and the illuminating assembly. A processing module controls the imaging assembly such that the camera acquires an image or images of the sample of a first field of view, and the image or images are automatically transmitted to a processing module and processed by the first processing module automatically in real-time based on a predefined criterion, so as to determine an interested region in the image and so as to obtain a coordinate information regarding to the interested region, and the coordinate information regarding to the interested region is transmitted to a processing module, where this module controls the illuminating assembly to illuminate the interested region of the sample according to the received coordinate information regarding to the interested region. It is therefore an object to process the proteins, lipids, nucleic acids, or biochemical species in an area of interest specified by fluorescent signals or structural signature of biological images. Additional object is to collect a large amount of proteins, lipids, and nucleic acids through high content labeling and purification in order to identify proteins, lipids, nucleic acids, or biochemical species of interest in the area of interest by analytical approaches including mass spectrometry and sequencing.