A simple extraction protocol and an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method for the determination of the syringyl/guaiacyl (S/G) ratio in lignin is reported herein. The method was entirely developed using stems of three Eucalyptus species, which were hydrolyzed with NaOH and partitioned with ethyl ether; vanillin (from the G monomer) and syringaldehyde (from S monomer) were quantified. The S/G ratios obtained were comparable to those usually reported for eucalyptus. The data for one of the eucalyptus species were compared with those obtained with a widely accepted method using thioacidolysis and gas chromatography-mass spectrometry (GC-MS). The method was also applied to sugarcane and showed to be reliable. The yield of the NaOH hydrolysis of the monolignols ranged from 89.94 to 95.69%, with more than 77.12% of recuperation in the liquid-liquid extraction. The whole analytical procedure was validated, achieving results with less than 4.38% of variation. The lowest LOD and LOQ were 0.01 and 0.05 μg/mL, respectively. In addition, the method combines reliability and a fast and direct quantification.