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Background and Objectives: Drug repurposing provides an opportunity to identify anticancer activities among established pharmacological agents. Labetalol (LB), an α₁- and non-selective β-adrenergic receptor antagonist, has been insufficiently investigated in melanoma. This study evaluated the potential anti-melanoma activity of LB in A375 human melanoma cells, compared its effects with those observed in HaCaT immortalized non-tumoral keratinocytes, and investigated the cellular mechanisms associated with LB-induced cytotoxicity. Materials and Methods: A375 and HaCaT cells were exposed to 75-500 μM LB for 24 h. Cell viability and lysosomal dye retention were assessed using MTT and NRU assays. A DMSO-only concentration series (0.075-0.50% v/v) was additionally evaluated by MTT in both cell lines. Mitochondrial membrane potential, mitochondrial staining patterns, nuclear morphology, cytoskeletal organization, caspase-3/7 and caspase-9 activities, and plasma membrane integrity were evaluated using JC-1, MitoTracker Red CMXRos, immunofluorescence, luminescence-based caspase assays, and AO/PI staining. The acute irritation potential of 500 μM LB was assessed using the HET-CAM assay. Results: DMSO alone did not significantly reduce viability over the investigated concentration range in either cell line. LB reduced A375 cell viability in a concentration-dependent manner, with a 24 h IC₅₀ of 422.5 μM and a viability of 33.61% at 500 μM. Under the same experimental conditions, HaCaT viability remained at 89.01% at 500 μM, and an IC₅₀ was not reached within the investigated concentration range. At the highest concentration, neutral red uptake and the JC-1 aggregate/monomer ratio decreased to approximately 33% and 24% of the corresponding control values, respectively. The apoptotic index increased from approximately 3% in control cells to 31%, while caspase-3/7 and caspase-9 activities increased to approximately 482% and 324% of the control. Qualitative imaging demonstrated mitochondrial staining redistribution, cytoskeletal disorganization, apoptosis-associated morphology, and loss of plasma membrane integrity at higher concentrations. In the HET-CAM assay, 500 μM LB produced a mean irritation score of 0.71 ± 0.27, within the non-irritant range. Conclusions: LB displayed concentration-dependent in vitro cytotoxicity in A375 melanoma cells, whereas HaCaT keratinocytes showed limited changes under the investigated conditions. LB treatment was also associated with mitochondria-associated apoptotic signaling in A375 cells. These findings provide preliminary evidence supporting further investigation of LB within a melanoma-directed drug-repurposing strategy. Additional studies are required to establish receptor dependence, achievable local exposure, safety, and translational relevance.