Brewer's spent grains (BSG) offer valuable opportunities for valorization beyond its conventional use as animal feed. Among its components, lignin-a natural polymer with inherent antioxidant properties-holds significant industrial potential. This work investigates the use of microwave-assisted extraction combined with acidic natural deep eutectic solvents (NaDESs) for efficient lignin recovery, evaluating three different NaDES formulations. The results indicate that choline chloride-lactic acid (ChCl-LA), a NaDES with superior thermal stability as confirmed via thermogravimetric analysis (TGA), is an ideal solvent for lignin extraction at 150 °C and 15 min, achieving a balance of high yield and quality. ChCl-LA also demonstrated good solubility and cell disruption capabilities, while microwaves significantly reduced processing time and severity. Under optimal conditions, i.e., 150 °C, 15 min, in the presence of ChCl-LA NaDES, the extracted lignin achieved a purity of up to 79% and demonstrated an IC50 (inhibitory concentration 50%) of approximately 0.022 mg/L, indicating a relatively strong antioxidant activity. Fourier transform infrared (FTIR) and 2D-HSQC NMR (heteronuclear single quantum coherence nuclear magnetic resonance) spectroscopy confirmed the successful isolation and preservation of its structural integrity. This study highlights the potential of BSG as a valuable lignocellulosic resource and underscores the effectiveness of acidic NaDESs combined with microwave extraction for lignin recovery.