The current research deals with the use of single-cell inductively coupled plasma-mass spectrometry (scICP-MS) for the assessment of titanium dioxide nanoparticle (TiO2NP) and silver nanoparticle (Ag NP) associations in cell lines derived from aquaculture species (sea bass, sea bream, and clams). The optimization studies have considered the avoidance of high dissolved background, multi-cell peak coincidence, and possible spectral interferences. Optimum operating conditions were found when using a dwell time of 50 μs for silver and 100 μs for titanium. The assessment of associated TiO2NPs by scICP-MS required the use of ammonia as a reaction gas (flow rate at 0.75 mL min−1) for interference-free titanium determinations (measurements at an m/z ratio of 131 from the48Ti(NH)(NH3)4adduct). The influence of other parameters such as the number of washing cycles and the cell concentration on accurate determinations by scICP-MS was also fully investigated. Cell exposure trials were performed using PVP-Ag NPs (15 and 100 nm, nominal diameter) and citrate-TiO2NPs (5, 25, and 45 nm, nominal diameter) at nominal concentrations of 10 and 50 μg mL−1for citrate-TiO2NPs and 5.0 and 50 μg mL−1for PVP-Ag NPs. Results have shown that citrate-TiO2NPs interact with the outer cell membranes, being quite low in the number of citrate-TiO2NPs that enters the cells (the high degree of aggregation is the main factor which leads to the aggregates being in the extracellular medium). In contrast, PVP-Ag NPs have been found to enter the cells.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1007/s00216-023-04723-6.