Accurate determination of lignin is essential to understand the mechanism by which it inhibits carbohydrate digestion. Available analytical procedures have repeatedly produced conflicting results. The gravimetric methods ADL, potassium permanganate lignin (PL) and KL were compared against the spectroscopic acetyl bromide lignin method (ABL). Grass species Brachiaria brizantha ‘Marandú’ and ‘Xaraés’, Panicum maximum ‘Mombaça’ and Pennisetum purpureum ‘Cameroon’ and ‘Napier’ were harvested at 7 maturity stages. A completely randomized experimental design with duplicate analysis for the lignin assays was used. A randomized block design was used for the in vitro experiment. Highly significant effects for maturity, lignin method and their interaction on lignin content were observed. The ADL yielded the lowest values (28.05 to 103.05 g/kg DM) in all species. Values for PL were in accordance to previous observations from our laboratory and other authors (65.45 to 160.65 g/kg DM). The KL values observed (61.35 to 136.20 g/kg DM) were approximately double the values observed for ADL. Values for ABL were higher than all corresponding values from the other methods. Strong negative correlations between lignin contents and IVDMD or IVNDFD were observed for all methods. Higher correlations were observed for IVDMD than for IVNDFD, contrary to what could be expected, because lignin affects the CW digestibility but not the cellular contents. Regression analysis of ADL, KL and ABL produced negative slopes when plotted against IVDMD and IVNDFD. The PL method failed to reliably estimate the digestibility of tropical grasses, possibly because of other substances (pectin, tannins or flavonoids) solubilized by the KMnO₄ solution. Although ADL and KL had similar correlations with digestibility and therefore, estimate digestibility of forages with similar accuracy, these methods use strong acids and require determination of ash in the lignin residues, increasing time and cost of analysis. The ABL method has no need for corrections and is a fast and a convenient method for determination of total lignin content in plants, thus, a good option for routine laboratory analysis.