The aim of this study was to evaluate marine algae extracts in terms of their anti-inflammatory activity using a combination of chromatographic separation and chemical detection with subsequent infrared vibrational spectroscopy identification. Extraction parameters, chemical fingerprint, and the activity levels were considered for the method optimization. High-performance thin-layer chromatography (HPTLC) combined with microchemical derivatization, was used to separate and detect bioactive compounds with antioxidant activity and anti-inflammatory activities, and to detect different classes of terpenoids. Infrared attenuated total reflectance (ATR) spectral analysis of the bands with bioactive compounds, identified sulfated polysaccharides to be responsible for the anti-inflammatory activity in extracts of brown algae Carpoglossum confluens and Phyllospora comosa. Steroids as unique antioxidants with significant free radical scavenging activities were observed in extracts of brown algae Cystophora platylobium, Cystophora retorta, Carpoglossum confluens and Phyllospora comosa. HPTLC combined with biochemical assays and FTIR-ATR spectrometry was demonstrated to be a straightforward strategy for bioprofiling marine algae extracts.