This study focused on the detection and quantification of organic micelle-type nanoparticles (NPs) with polysorbate components (polysorbate 20 and polysorbate 80) in their micelle shells that could be used to load biologically active compounds into fruit juice. Several advanced analytical techniques were applied in the stepwise method development strategy used. In the first phase, a system consisting of ultrahigh-performance liquid chromatography employing a size exclusion column coupled with an evaporative light scattering detector (UHPLC-SEC-ELSD) was used for the fractionation of micelle assemblies from other, lower molecular weight sample components. The limit of detection (LoD) of these polysorbate micelles in spiked apple juice was 500 μg mL(-1). After this screening step, mass spectrometric (MS) detection was utilized to confirm the presence of polysorbates in the detected micelles. Two alternative MS techniques were tested: (i) ambient high-resolution mass spectrometry employing a direct analysis in real time ion source coupled with an Orbitrap MS analyzer (DART-Orbitrap MS) enabled fast and simple detection of the polysorbates present in the samples, with a lowest calibration level (LCL) of 1000 μg mL(-1); (ii) ultrahigh-performance reversed-phase liquid chromatography coupled with high-resolution time-of-flight mass spectrometry (UHPLC-HRTOF-MS) provided highly selective and sensitive detection and quantification of polysorbates with an LCL of 0.5 μg mL(-1).