With the likely release of engineered nanoparticles into the aquatic environment, developing appropriate analytical methods for occurrence surveys has become a priority. To this end, a method towards the quantification of silver nanoparticles (AgNP) in lake water samples using asymmetric flow field flow fractionation (AF4) and inductively coupled plasma mass spectrometry (ICP-MS) is reported. Therefore, light scattering (LS) detection was used to develop an assessment metric to evaluate relative accuracy among AF4 separations. This assessment metric is applied to optimization of cross flow (Vx) protocols in AF4 separation interfaced with LS detection using mixtures of polystyrene beads. AF4 has several instrumental parameters that may have a direct effect on separation performance. A sensitivity analysis utilizing orthogonal factional factorial design and graphical analysis was applied to ascertain the relative importance of five AF4 primary/instrumental factor settings when analyzing synthetic freshwaters containing AgNPs. The most important and significant AF4 primary/instrumental factors were buffer concentration and Vx velocity, while the least impacting was Vx ramp time. Optimal settings were also generated for each of the factors within the range of settings explored. A parallel orthogonal fractional factorial design was employed to evaluate the effects of five environmental factors, or water quality characteristics, on the separation. None of these water quality characteristic effects or interactions were found to be significant. Finally, the developed methodology was applied towards AgNP quantitation in a natural lake water sample using AF4 followed by online inductively coupled plasma mass spectrometry (ICP-MS). The impacts of various AF4 system components and natural organic matter (NOM) on AgNP quantitation were explored. Isotope enriched Ag ions (Ag+) were used to identify Ag speciation following AF4 separation. Ag quantitation was achieved within 10% of a spiked “challenge” concentration of AgNP in lake water using the standard addition method to compensate for natural matrix and system complexity. Further investigations into potential Ag+-NOM and AgNP-NOM interactions were performed using fluorescence spectroscopy. The corresponding results suggest that these interactions affect the molar mass and physical conformation of the NOM particles in the sample. Advisors/Committee Members: Galyean, Anne, Weinberg, Howard, Vreeland, Wyatt, Leopold, Michael, Cory, Rose, Coronell, Orlando.