Lysosomes are acidic organelles responsible for lipid catabolism, and their functions can be disrupted by cationic amphiphilic drugs that neutralize lumenal pH and thereby inhibit most lysosomal hydrolases. These drugs can also induce lysosomal membrane permeabilization and cancer cell death, but the underlying mechanism remains elusive. Here, we uncover that the cationic amphiphilic drugs induce a substantial accumulation of cytolytic lysoglycerophospholipids within the lysosomes of cancer cells, and thereby prevent the recycling of lysoglycerophospholipids to produce common membrane glycerophospholipids. Using quantitative mass spectrometry-based shotgun lipidomics, we demonstrate that structurally diverse cationic amphiphilic drugs, along with other types of lysosomal pH-neutralizing reagents, elevate the amounts of lysoglycerophospholipids in MCF7 breast carcinoma cells. Lysoglycerophospholipids constitute ∼11 mol% of total glycerophospholipids in lysosomes purified from MCF7 cells, compared with ∼1 mol% in the cell lysates. Treatment with cationic amphiphilic drug siramesine further elevates the lysosomal lysoglycerophospholipid content to ∼24 mol% of total glycerophospholipids. Exogenously added traceable lysophosphatidylcholine is rapidly acylated to form diacylphosphatidylcholine, but siramesine treatment sequesters the lysophosphatidylcholine in the lysosomes and prevents it from undergoing acylation. These findings shed light on the unexplored role of lysosomes in the recycling of lysoglycerophospholipids and uncover the mechanism of action of promising anticancer agents.