Abstract The amount of hydroxyl groups, particularly phenolic, is one of the most important parameters in lignins, as it is an indicator of lignin reactivity. Ultraviolet (UV) Spectrophotometry is a simple and inexpensive method for determining phenolic hydroxyls in lignin. Ionization Difference Ultraviolet Spectrophotometry (Δε-method) relies on the analysis of solubilized lignin at neutral and alkaline conditions with a UV spectrophotometer. We added a slope analysis to the ∆ε-method and dubbed the resulting method ∆ε-IDUS (Ionization Difference UV Spectrophotometry). We assessed the reliability of ∆ε-IDUS by studying the well-known Indulin AT lignin. Additionally, ∆ε-IDUS was applied to a previously uncharacterized milox lignin. When compared to ¹³C-NMR, ∆ε-IDUS underestimated the amount of phenolic hydroxyls for Indulin AT, possibly due to neglecting second phenolic hydroxyls in some lignin units, which resist ionization because of steric hindrance. Nevertheless, the results agreed with previously reported values and confirm that ∆ε-IDUS is useful to screen lignins based on their phenolic hydroxyl group content.