Autophagy refers to the catabolic process in eukaryotic cells that delivers cytoplasmic material to lysosomes for degradation. This highly conserved process is involved in the clearance of long-lived proteins and damaged organelles. Consequently, autophagy is important in providing nutrients to maintain cellular function under starvation, maintaining cellular homeostasis, and promoting cell survival under certain conditions. Several pathways, including mTOR, have been shown to regulate autophagy. However, the impact of lysosomal function impairment on the autophagy process has not been fully explored. Basic lipophilic compounds can accumulate in lysosomes via pH partitioning leading to perturbation of lysosomal function. Our hypothesis is that these types of compounds can disturb the autophagy process. Eleven drugs previously shown to accumulate in lysosomes were selected and evaluated for their effects on cytotoxicity and autophagy using ATP depletion and LC3 assessment, respectively. All eleven drugs induced increased staining of endogenous LC3 and exogenous GFP-LC3, even at non toxic dose levels. In addition, an increase in the abundance of SQSTM1/p62 by all tested compounds denotes that the increase in LC3 is due to autophagy perturbation rather than enhancement. Furthermore, the gene expression profile resulting from in vitro treatment with these drugs revealed the suppression of plentiful long-lived proteins, including structural cytoskeletal and associated proteins, and extracellular matrix proteins. This finding indicates a retardation of protein turnover which further supports the notion of autophagy inhibition. Interestingly, upregulation of genes containing antioxidant response elements, e.g. glutathione S transferase and NAD(P)H dehydrogenase quinone 1 was observed, suggesting activation of Nrf2 transcription factor. These gene expression changes could be related to an increase in SQSTM1/p62 resulting from autophagy deficiency. In summary, our data indicate that lysosomal accumulation due to the basic lipophilic nature of xenobiotics could be a general mechanism contributing to the perturbation of the autophagy process.