Experimental design methodology was used to optimize the HPLC separation of various relevant phenolic acids from an artificial mixture. The effect of four characteristic factors of the HPLC procedure on the Chromatographic Response Function was investigated by a Central Composite Face-Centred Design and Multi Linear Regression fitting. The eight phenolic acids were successfully separated. The optimal HPLC setting condition was applied to rapid screening of free phenolic acids from
Striga hermonthica
(Del.) Benth. (Scrophulariaceae),
Guiera senegalensis
J. F. Gmel (Combretaceae) and unifloral honey from
Acacia sp. (Mimosaceae). None of reference phenolic acid was identified in
S. hermonthica extract when some of them were detected in
G. senegalensis and honey extracts. This work is considered as a first step for a largest phenolic acids screening from various preparations used in Burkina Faso folk medicine to justify some of their properties or potentialities.