The replacement of synthetic dyes has gained increasing attention due to stricter regulatory policies and growing health concerns. Microbial carotenoids represent a promising alternative to artificial food colorants; however, their large-scale production is limited by the high cost of raw materials. In this context, the valorization of lignocellulosic biomass offers a strategy to develop low-cost substrates for microbial bioprocesses. Sotol bagasse (SB), an underutilized lignocellulosic residue generated during sotol production, was composed of 24% cellulose, 14% hemicellulose, and 42% lignin. A non-thermal plasma pretreatment, optimized through response surface methodology, achieved up to 29% of lignin removal. Subsequent enzymatic hydrolysis yielded a total sugar concentration of 28 g/L. The resulting hydrolysate supported the growth of Rhodotorula glutinis, yielding 4.4 g/L of biomass and 0.91 mg/L of carotenoids. To the best of our knowledge, this is the first report describing the use of non-thermal plasma as a pretreatment strategy for sotol bagasse, demonstrating its potential as a chemical-free approach for lignocellulosic valorization and sustainable microbial carotenoid production.