Photoaffinity labeling (PAL) has been widely applied in various research areas such as medicinal chemistry, chemical biology and structural biology, owing to its capability of investigating non-covalent ligand-protein interactions under native environments and elucidating protein structures, functions etc. One important application of this technique is to use affinity-based proteome profiling (AfBP) coupled with bioimaging for profiling drug-target interactions in situ. In order to accurately report drug-target interactions via these approaches, several considerations as follows need to be made: (1) maximally retaining bioactivities of photoprobes upon functionalization with a photoreactive group and a reporter tag from a parental compound; (2) performing proteome profiling and imaging in situ simultaneously, to monitor drug-target interactions in different manners; and (3) developing excellent photo-crosslinkers capable of photo-crosslinking and fluorescence turn-on at the same time. With these considerations in mind, we have developed three versions of "minimalist" bioorthogonal handle-containing photo-crosslinkers (L3-L6) during the years and successfully applied them in all kinds of small bioactive molecules for protein labeling and cellular imaging studies. In this mini-review, the features and functions of these linkers are specifically highlighted and summarized.