Astrocytes play a critical role in the onset and progression of amyotrophic lateral sclerosis (ALS), a fatal disorder marked by the rapid degeneration of motor neurons (MNs) in the central nervous system. Despite evidence that ALS astrocytes are toxic to MNs, the earliest pathological changes leading to their neurotoxic phenotype remain unclear. In this study, we generated human astrocytes from induced pluripotent stem cells (iPSCs) of patients with ALS-associated A4V mutation in superoxide dismutase 1 (SOD1) and controls to investigate early disease-related protein alterations. Proteomic analysis revealed significant changes in proteins previously linked to ALS pathology. Notably, ALS astrocytes exhibit an increment of proteins associated with an aberrant activation of the innate immune system. The proteome profile of ALS astrocytes transduced with AAV particles encoding fibroblast growth factor 4 (FGF4) was largely reversed to resemble that of control astrocytes, suggesting a reversion of the pathological protein profile. However, trying to reduce astrocyte reactivity with AAV-FGF4 in vivo fails to prevent MN death in ALS. To investigate the underlying potential mechanism behind the treatment failure, we evaluate the response of ALS and control astrocytes in a pro-inflammatory environment. Our analysis indicated that ALS astrocytes show an exacerbated expression of proteins related to immune reactivity, indicating that TNFα could abrogated the rescuing effects of FGF4 on ALS astrocytes. Our data demonstrate that ALS astrocytes sustain elevated levels of proteins related to the cGAS-STING pathway, such as XRCC5/XRCC6, and scaffold elements for IRF3 phosphorylation after TNFa+FGF4 treatment. In conclusion, this study shows the potential of the patient-derived iPSC models using proteomics as molecular readout to capture the cell-autonomous contribution of astrocytes in the pathogenesis of ALS and to test new therapeutic approaches to fine-tune the responsiveness of astrocytes and their contribution to neuroinflammation in ALS.