Protein formulations may contain subvisible particle (SbVP) impurities that can vary (e.g., in number, size, shape, density, refractive index and transparency) depending on the formulation composition, environmental stresses and the type of protein. Additionally formulation solutions may differ in their physical properties including turbidity, color, viscosity, density and refractive index. This study examined the impact of these formulation matrix parameters on the ability to size and count subvisible particles using a variety of analytical methods including two light obscuration (HIAC, Syringe) and two digital imaging instruments (MFI®, FlowCAM®). Several subvisible particle standards were tested, including polystyrene and glass beads as well as a new pseudo-protein particle standard, in order to also study of the effect of subvisible particles with different properties.