<p>Accurate quantification of chitin oligosaccharides (COS) is essential for enzymatic hydrolysis research and bioactivity exploration, yet current analytical methods lack harmonization and limit profiling and quantitative accuracy. This study addresses these challenges by optimizing a gas chromatography (GC) method, validated to achieve precise quantification of COS in the range of DP1-DP4. By optimizing the silylation protocol, we increased the signal-to-noise (S/N) ratio in all DP standards, including a sevenfold increase in the S/N ratio of DP4. This GC method demonstrated robust linearity (<i>R</i>² ≥ 0.99), high sensitivity (DP1 range from 10 to 2000&#xa0;µg&#xa0;mL⁻¹), strong precision (&lt; 15% coefficient variation), high accuracy (recoveries 90–100%) and repeatability (&lt; 10% coefficient variation) with COS standards. Additionally, a quantitative high-performance thin-layer chromatography (HPTLC) method for COS DP1-DP6 was validated, offering a rapid and cost-effective alternative. A comparative analysis was done using real-world samples produced from a novel endochitinase from <i>Thermothelomyces thermophilus</i> applying the two novel validated methods, high-performance liquid chromatography (HPLC) and a non-specific reducing sugar assay (DNS). Both novel methods demonstrated agreement in the ratio of COS products generated, which ranged from DP1 to DP3, and in the theoretical degree of hydrolysis reached (96 ± 9.2% and 95 ± 3.3%, for GC and HPTLC, respectively), while the conventional methods reported a lower product yield of 75 ± 1.2% with HPLC and 53 ± 1.0% with DNS. This analytical toolkit enhances low-DP COS characterization and facilitates more accurate in chitin hydrolysis research.</p> Graphical Abstract <p></p>