This study aimed to develop HPTLC fingerprint profile and identification of antioxidant molecules from active extract and fractions of Ambrosia maritima and Ammi majus using ultra performance liquid chromatography-mass spectroscopy (UPLC-MS). The antioxidant activity evaluated by using DPPH (1,1-diphenyl-2-picryl hydrazyl) method and HPTLC fingerprinting were carried out using CAMAG HPTLC system equipped with Linomat IV applicator, TLC scanner III, Reprostar 3, Camag twin through glass tank for development, and Wincasts1.2.3. The methanolic extract of A. maritima and A. majus shows highest antioxidant radical scavenging activity (87 and 58%, respectively). The ethyl acetate, aqueous fractions of A. maritima and aqueous fraction of A. majus shows the highest antioxidant activity (86, 82 and 81% respectively). The HPTLC profile of A. maritima ethyl acetate fraction indicates presence of nineteen compounds, ellagic (0.61%) and gallic phenolic acids (0.54%) content, respectively and ten compounds have been detected. The HPTLC profile of A. majus aqueous fraction indicate presence of twelve compounds, ellagic acid content (0.79%) and six compounds were detected. The HPTLC profile of A. maritime aqueous fraction indicate presence of nine compounds, ellagic acid content (2.54%) and eight compounds have been detected by UPLC-MS analysis. Key words: Ambrosia maritima, Ammi majus, Antioxidant, HPTLC fingerprint, UPLC-MS analysis.