Metabolic dysfunction-associated fatty liver disease (MAFLD) encompasses a spectrum of progressive liver disorders that, if unresolved, can trigger inflammation and fibrosis. Hepatic stellate cells (HSCs) are key mediators of these fibrotic processes in the liver. In healthy tissue they store large amounts of lipids, primarily retinol esters (RE) and triacylglycerols (TAGs) in intracellular lipid droplets (LDs). Upon liver injury or chronic inflammation quiescent HSCs transdifferentiate into “activated” myofibroblast-like cells. Hallmark features of activated HSCs include increased proliferative capacity, directed migration towards injury sites and increased secretion of extracellular matrix proteins (scar tissue). However, the most prominent change is the shift in LD phenotype from large, RE-rich droplets to more dynamic, smaller LDs enriched in polyunsaturated fatty acid (PUFA)-containing TAGs. Understanding the mechanisms involved in HSC transdifferentiating is crucial for targeting liver fibrosis and its progression to more severe conditions. To address this, we compared proteomes of pseudo-quiescent and activated LX-2 cells (an immortalized human HSC line) by label-free quantitative (LFQ) proteomics and evaluated serine hydrolase activity via activity-based protein profiling (ABPP) to probe metabolic pathway alterations. The diverse enzyme superfamily of serine hydrolases encompasses many key lipases likely involved in LD remodelling. To dissect the specific contribution of neutral lipolysis, we pharmacologically inhibited ATGL – the rate-limiting enzyme of the lipolytic cascade – and assessed its effects on both pseudo-quiescent and activated HSCs. To date, our results demonstrate that while the protein level of key serine hydrolases (e.g., ATGL) remained unchanged, their enzymatic activities were significantly modulated during activation. Inhibition of ATGL abrogated LD remodelling, underscoring its essential role in this process. In contrast, overall HSC activation was only modestly affected by impaired lipid hydrolysis, suggesting that LD remodelling may be a consequence rather than a prerequisite of HSC activation. Our findings elucidate ATGL’s pivotal function in LD dynamics during HSC activation and offer new insights into our understanding of lipid hydrolysis in HSC activation.