Currently, the interest in microalgae as a source of biologically active components exploitable as supplementary ingredients to food/feed or in cosmetics continues to increase. Existing research mainly aims to focus on revealing and recovering the rare, cost competitive components of the algae metabolom. Because these components could be of very different physicochemical character, a universal approach for their isolation and characterization should be developed. This study demonstrates the systematic development of the extraction strategy that represents one of the key challenges in effective algae bioprospecting, which predefines their further industrial application. By using of Trachydiscus minutus as a model microalgae biomass, following procedures were tested and critically evaluated in order to develop the generic procedure for microalgae bioprospecting: (i) various ways of mechanical disintegration of algae cells enabling maximum extraction efficiency, (ii) the use of a wide range of extraction solvents/solvent mixtures suitable for optimal extraction yields of polar, medium-polar, and non-polar compounds, (iii) the use of consecutive extractions as a fractionation approach. Within the study, targeted screening of selected compounds representing broad range of polarities was realized by ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometric detection (UHPLC-HRMS/MS), to assess the effectiveness of undertaken isolation steps. As a result, simple and high-throughput extraction-fractionation strategy based on consecutive extraction with water-aqueous methanol-hexane/isopropanol was developed. Moreover, to demonstrate the potential of the UHPLC-HRMS/MS for the retrospective non-target screening and compounds identification, the collected mass spectra have been evaluated to characterize the pattern of extracted metabolites. Attention was focused on medium-/non-polar extracts and characterization of lipid species present in the T. minutus algae. Such detailed information on the composition of native (non-hydrolyzed) lipids of this microalga has not been published yet.