The leaves of the pantropical genus Bauhinia (Fabaceae) are popularly known as cow's-paw or cow's hoof due to their unique characteristic bilobed aspect. The species Bauhinia forficata (Brazilian Orchid-tree) is widely used in folk medicine as an antidiabetic. This article deals with the quantitative analysis of kaempferitrin from B. forficata medicinal extract (aqueous and hydro alcoholic) using the LC method, and the comparison of kaempferitrin content in leaves collected from two different regions in the south Brazil. The total flavonoid content assessed by LC was also compared with the classical spectrophotometric determination. Kaempferitrin was found in different amounts, in samples from two geographical areas (Telêmaco Borba/PR and Itajaí/SC), for aqueous (368.68 and 77.91 μg/mL) and hydro alcoholic extracts (1952.59 and 211.61 μg/mL), respectively. The method was subjected to recovery assay, to determine its accuracy. A marked difference in total flavonoid concentration was observed in relation to kaempferitrin content: 2759.95 and 2188.20 μg/mL for the fluidextract and 863.35 and 856.77 μg/mL for the aqueous extract (Telêmaco Borba/PR and Itajaí/SC). The spectrophotometric assay overestimated the total flavonoid content (3620 μg/mL) in relation to the LC assay.