Luminescent samarium (Sm) and europium (Eu) doped silica nanoparticles (SiO₂ NPs) were investigated for use in multiple target immunoassays. Particles with a diameter of 40 ± 10 nm were synthesized and applied to multiplexed immunoassays in a single well of an assay plate. The luminescent behavior of the lanthanide doped NPs was compared to conventional dyes in solution and in surface confined model immunoassays against two analytes. Luminescence from the dyes and NPs was detected with an imaging spectroscopic system and this could be processed with a simple least squares procedure. In solution, NPs exhibited linear mixing behaviour while conventional fluorescent dyes required careful control to solution conditions, particularly pH. A conventional dye system consisting of FITC and Cy3 applied to a two analyte model system resulted in up to 70% energy transfer from FITC to Cy3 under conditions of high surface coverage. This made quantitative analysis intractable for unknown samples. The lanthanide doped silica nanoparticles responded well to both analytes (<5% relative std. dev.). The results demonstrate that NPs provide alternatives to conventional dyes due to low susceptibility to pH and related solution conditions, absence of energy transfer, single excitation wavelength range, and high stokes shift. These properties allowed quantitative analysis of multiple analytes in the same well.