When investigating the potential use of plants as a raw material for an all-natural cosmetic formulation, the main parameters are the chemical composition, antioxidant potential, antimicrobial action, and toxicity. Additionally, the production of natural cosmetics should also consider the availability of primary materials and the environmental and socioeconomic impact. Gliricidia sepium is a species that produces a large amount of plant material, being cultivated in the agroforestry system. However, studies of phytochemical composition and chemical spatial distribution are scarcely using the MALDI MS (matrix-assisted laser desorption ionization mass spectrometry) and MALDI MSI (mass spectrometry imaging) techniques. A methodology was developed to optimize ionization parameters and analysis conditions by evaluating the efficiency of three matrices: α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid (DHB), and 2-mercaptobenzothiazole in MALDI MS analysis. All results were compared to ESI MS (electrospray ionization mass spectrometry), and afterward, MALDI MSI analysis was performed on the leaf surface. This study showed through phytochemical analysis that G. sepium leaves are composed of polyphenols and tannins, concluding that the methanolic extract had a higher amount of flavonoid content. Four compounds were identified on the leaf surface, and their spatial distribution was analyzed by MALDI MS using DHB as a matrix. Kaempferol, isorhamnetin, and some fatty acids showed potential applicability for cosmetical use. All the extracts presented antioxidant activity or antimicrobial action and no cytotoxicity. Therefore, extracts of G. sepium could be used as raw materials in cosmetics.