The release of nanoplastics (NP) from the weathering and degradation of plastics represents one of the major concerns for the environment given their pervasiveness in cells. A methodology for the detection of anisotropic changes induced by polystyrene NP is proposed using fluorescence polarization. The commercially available probe fluorescein octadecyl ester (FOE) has the property to interact in hydrophobic environments (phospholipids) which could be measured by fluorescence polarization spectroscopy. Although increasing the concentrations of 50 nm polystyrene NP in buffer alone did not change polarization, the addition of the subcellular fraction increased polarisation of the dye in a concentration-dependent manner. The assay was performed in mussels exposed to primary-treated effluents and revealed increased anisotropy of the subcellular fraction of the digestive gland suggesting NP-like effects. Although lipid contents were significantly correlated with fluorescence polarization (r = 0.65; p < 0.001), the increase in polarization was not entirely explained by changes in lipid droplets. In conclusion, polystyrene NP induces anisotropic effects at the subcellular fraction of the digestive gland as determined with the FOE probe. Mussels exposed to primary-treated effluents displayed NP-like anisotropic effects suggesting that these effluents contain NPs.