Nanomedicines are often recognized by the innate immune system as a threat, leading to unwanted clearance due to complement activation. This adverse reaction not only alters the bioavailability of the therapeutic but can also cause cardiopulmonary complications and death in a portion of the population. There is a need for tools for assessing complement response in the early stage of development of nanomedicines. Currently, quantifying complement-mediated response in vitro is limited due to differences between in vitro and in vivo responses for the same precursors, differences in the complement systems in different species, and lack of highly sensitive tools for quantifying the changes. Hence, we have worked on developing complement assay conditions and sample preparation techniques that can be highly sensitive in assessing the complement-mediated response in vitro mimicking the in vivo activity. We are screening the impact of incubation time, nanoparticle dosage, anticoagulants, and species of the donor in both blood and blood components. We have validated the optimal assay conditions by replicating the impact of zeta potential seen in vivo on complement activation in vitro. As observed in our previous in vivo studies, where nanoparticles with neutral zeta-potential were able to suppress complement response, the change in the complement biomarker was least for the neutral nanoparticles as well through our developed guidelines. These assay conditions provide a vital tool for assessing the safety of intravenously administered nanomedicines.