Lysosomal biogenesis is an essential adaptive process by which lysosomes exert their function in maintaining cellular homeostasis. Defects in lysosomal enzymes and functions lead to lysosome-related diseases, including lysosomal storage diseases and neurodegenerative disorders. Thus, activation of the autophagy-lysosomal pathway, especially induction of lysosomal biogenesis, might be an effective strategy for the treatment of lysosome-related diseases. In this study, we established a lysosome-based screening system to identify active compounds from natural products that could promote lysosomal biogenesis. The subcellular localizations of master transcriptional regulators of lysosomal genes, TFEB, TFE3 and ZKSCAN3 were examined to reveal the potential mechanisms. More than 200 compounds were screened, and we found that Hdj-23, a triterpene isolated from Walsura cochinchinensis, induced lysosomal biogenesis via activation of TFEB/TFE3. In summary, this study introduced a lysosome-based live cell screening strategy to identify bioactive compounds that promote lysosomal biogenesis, which would provide potential candidate enhancers of lysosomal biogenesis and novel insight for treating lysosome-related diseases.© 2022 The Author(s).