Complement, a crucial component of humoral innate immunity, plays a pivotal role in opsonizing nanopharmaceuticals and pathogens through the third complement protein (C3) while concurrently releasing proinflammatory factors such as C3a and C5a (Moghimi and Farhangrazi, 2013; Moghimi et al., 2020). Several approaches exist for measuring complement activation, including by hemolysis of sensitized sheep red blood cells (CH50) and measurements of pathway-specific and terminal activation markers such as Bb, C3a, iC3b, C3bc, C4a, C5a, and sC5b-9 by enzyme-linked immunosorbent assay (ELISA) (Kirschfink and Mollnes, 2003). At the same time, several groups, including ours, assessed the level of C3 on various surfaces using flow cytometry and fluorescent microscopy, for example, on bacteria (Pathak et al., 2018), polymer beads (Rokstad et al., 2011), erythrocytes (Lam et al., 2021), and nanoparticles (Li et al., 2024). The disadvantage of these assays is that they are not entirely quantitative, are labor-intensive, and are particularly challenging to apply to nanosized particles. However, there is a clear need to measure the level of deposited C3 in multiple donors and nanoparticle types, as C3b/iC3b is the main opsonin aiding nanoparticle ingestion by phagocytic cells, and its disposition could also be correlated with other components of the protein corona. Dot blot immunoassays (DBI) are commonly employed to rapidly screen samples, including proteins and nucleic acids (Farrell, 2005), due to their simplicity and efficiency. DBI is especially useful when dealing with arrays of samples or when the quantity of sample material is limited (Renart et al., 1996). We developed a DBI to measure C3 deposition on nanoparticles (Li et al., 2024; Li et al., 2021; Benasutti et al., 2017), capitalizing on the sedimentation behavior of nanoparticles and liposomes under high g-forces. We successfully quantified C3 deposition among human donors and animal strains, measured the impact of complement regulators (IC50 value) on diverse nanomaterials, and determined the number of deposited proteins per nanoparticle (Li et al., 2024; Li et al., 2021; Vu et al., 2019). Here, the repeatability of DBI is validated, and C3 deposition is correlated with other complement activation markers measured in the fluid phase.