Lysosomes are specialized organelles that sense and respond to cellular metabolic status as well as recycle macromolecules and cell components. Given that lysosomal dysfunction is implicated in a wide range of disorders, it is imperative to examine their molecular contents. Nonetheless, the characterization of the lysosomal molecular contents remains a significant challenge due to the small fractional volume occupied by these organelles within a cell. To overcome these challenges, we generated a transgenic mouse model with (HA)-tagged lysosomes enabling rapid immunopurification of intact lysosomes (LysoIP) for multi-omic measurement of their content. To test the application of our model in understanding lysosomal dysfunction in human disorders, we conducted a multi-omic analysis of brain lysosomes obtained from mouse models of Batten disease, a neurodegenerative disorder caused by mutations in CLN3, which encodes a lysosomal transmembrane protein of unknown function. Loss of CLN3 caused an excessive buildup of glycerophosphodiesters (GPDs), which are byproducts of phospholipid breakdown. Our further studies show that CLN3 is required for the lysosomal egress of GPDs. We believe that the LysoIP workflow and its compatibility with multi-omic analyses will further elucidate the complex functions of lysosomes in disease and physiology.