Lysosomes are implicated in a wide spectrum of human diseases including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration and cancer. Profiling lysosomal content using tag-based lysosomal immunoisolation (LysoTagIP) in cell and animal models allowed major discoveries in the field, however, studying lysosomal dysfunction in human patients remains a challenge. Here, we report the development of the tagless LysoIP method to enable rapid enrichment of lysosomes, via immunoisolation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines. Isolated lysosomes are intact and suitable for subsequent multimodal omics analyses. To validate the utility of our approach, we employed the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells (PBMCs) derived from fresh blood of patients with CLN3 Batten disease, a neurodegenerative LSD. Metabolic profiling of isolated lysosomes showed massive accumulation of glycerophosphodiesters (GPDs) in patients’ lysosomes. Interestingly, a patient with a milder phenotype and genotype, displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify novel biomarkers and discover human-relevant disease mechanisms.