In this protocol, we describe the use of ChipCytometry to combine RNA hybridization and antibody staining for multiplexed tissue imaging of human formalin-fixed and paraffin-embedded tissue samples. The advantages of ChipCytometry are long-term storage for re-interrogation and advanced image quality by high dynamic range imaging of staining and background. A titrated pretreatment of tissue samples bypasses challenges because of the retrieval of antigens on coverslips and achieves an optimal staining quality at the minimal expense of tissue integrity. For complete details on the use and execution of this protocol, please refer to Jarosch et al. (2021).