Antibodies engage with the immune system and signal for effector functions through weak interactions between the conserved Fc region of the antibody and Fc receptors on the surfaces of immune cells. Additionally, the Fc region interacts with the neonatal Fc receptor (FcRn), which prolongs antibody circulation in blood. Here we utilized immobilized receptors (FcγRIIIa and FcRn) on affinity LC columns with online MS detection to examine the impact of antibody sequence mutations, PTMs and chemical modifications on receptor binding. The primary factors contributing to FcγRIIIa affinity were found to be IgG subtype and Fc glycosylation. Fc glycans with increasing levels of terminal galactose, and core afucosylation had the strongest binding affinity for FcγRIIIa. The overall elution trends for most species were consistent with published literature values, however some exceptions were observed. These deviations may be due to the use of immobilized aglycosylated FcγRIIIa receptor, highlighting that both antibody and receptor glycosylation are important for biological activity. Heat stressed affinity fractions were collected and subjected to LC-MS/MS peptide mapping to identify chemical modifications negatively impacting FcγRIIIa affinity (i.e enriched in low affinity fractions). FcRn affinity chromatography was also utilized to monitor attributes that impact FcRn binding and serum half-life based on their retention times. Chemical modifications such as Met oxidation on residues near the FcRn binding domain were observed in early eluting peaks (decreased affinity), and engineered sequence mutations to these residues were observed to elute later (increased affinity). By monitoring changes in retention time and using intact antibody masses for identification, changes in binding interactions can be directly mapped to specific modifications and residues. These observed retention time shifts were also found to correlate well with changes in binding affinity measured by an orthogonal AlphaLISA binding assay, providing reference points to calibrate and give context to the affinity chromatograms.