This paper reports a rapid HILIC-ESI–MS assay to quantify dipalmitoylphosphatidylcholine (DPPC) as component of lung surfactant for nanosafety studies. The technique was used to investigate the concentration-dependent sorption of DPPC to two-sizes of amorphous SiO2 nanoparticles (SiO2-NPs) in a MeOH:H2O (50/50 v/v) mixture and in cell culture medium. In MeOH:H2O (50/50 v/v), the sorption of DPPC was positively correlated with the nanoparticles concentration. A substantial affinity of small amorphous SiO2-NPs (25 nm) to DPPC standard solution compared to bigger SiO2-NPs (75 nm) was not confirmed for biological specimens. After dispersion of SiO2-NPs in DPPC containing cell culture medium, the capacity of the SiO2-NPs to bind DPPC was reduced in comparison to a mixture of MeOH:H2O (50/50 v/v) regardless from the nanoparticles size. Furthermore, HILIC-ESI–MS revealed that A549 cells internalized DPPC during growth in serum containing medium complemented with DPPC. This finding was in a good agreement with the potential of alveolar type II cells to recycle surfactant components. Binding of lipids present in the cell culture medium to amorphous SiO2-NPs was supported by means of HILIC-ESI–MS, TEM and ICP-MS independently.