Lysosomes are membrane-bound organelles that regulate protein degradation and cellular organelle recycling. Homeostatic alteration by lysosomotropic compounds has been suggested as a potential approach for the treatment of cancer. However, because of the high false-negative rate resulting from strong fluorescent background noise, few luminescent high-throughput screening methods for lysosomotropic compounds have been developed for cancer therapy. Imidazole is a five-membered heterocycle that can act within the acidic interior of lysosomes. To develop an efficient lysosomotropic compound screening system, we introduced an imidazole group to iridium-based complexes and designed a long-lifetime lysosomal probe to monitor lysosomal activity in living cells. By integrating time-resolved emission spectroscopy (TRES) with the novel iridium-based lysosomal probe, a high-throughput screening platform capable of overcoming background fluorescent interference in living cells was developed for discovering lysosomotropic drugs. As a proof-of-concept, 400 FDA/EMA-approved drugs were screened using the TRES system, revealing five compounds as potential lysosomotropic agents. Significantly, the most promising potent lysosomotropic compound (mitoxantrone) identified in this work would have showed less activity if screened using a commercial lysosomal probe because of interference from the intrinsic fluorescence of mitoxantrone. We anticipate that this TRES-based high-throughput screening system could facilitate the development of more lysosomotropic drugs by avoiding false results arising from the intrinsic fluorescence of both bioactive compounds and/or the cell background.