In virology-related studies, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is routine to inactivate body fluid samples carrying the virus to reduce the spread of the virus and guarantee the safety of biobankers and researchers. However, inactivation treatments may affect the molecular structure of proteins in biological samples, and it is necessary to select an inactivation method that has the least impact on the target molecule associated with protein detection techniques. Cytometric Bead Array (CBA), a novel and powerful technology, allows the simultaneous quantification of up to 10–30 different soluble proteins from one sample, with a particular focus on various cytokines and chemokines in human body fluids. But only a few studies have investigated the effect of inactivation methods on relevant assays. Therefore, this study aims to investigate various viral inactivation methods and evaluate their impact on LEGENDplex™ bead-based immunoassays. By detecting eight plasma samples and eight ascites samples, we assessed the impacts of heat denaturation, γ-irradiation, and paraformaldehyde (PFA) inactivation methods on certain protein components in plasma and ascites by LEGENDplex™ bead-based immunoassays. The results showed that heat treatment and γ-irradiation treatment had little effect on LEGENDplex™ bead-based immunoassays in both blood and ascites, while PFA treatment resulted in changes in multiple cytokines and chemokines. Among twenty-six cytokines and chemokines, perforin, I-TAC (CXCL11), Eotaxin (CCL11), and MIP-3α (CCL20) were more vulnerable to heat denaturation, γ-irradiation, and PFA treatments. To sum up, our findings provide evidence to inform the selection of optimal inactivation methods for reliable cytokine profiling in SARS-CoV-2-infected samples.