The conjugation of proteins to the outer membranes of liposomes is a standard procedure used in bioanalytical and drug delivery approaches. Herein, we describe the development of a liposome-based surrogate assay for the quantification of SARS-CoV-2 neutralizing antibodies. Taking into consideration differences in amino acid sequences within the receptor-binding domain (RBD) of SARS-CoV-2 Spike proteins derived from five selected variants of concern (VoC), we studied the impact of coupling chemistries on physicochemical properties and antigenicity. Naturally occurring lysine residues were used for standard EDC/NHS chemistry, while an N-terminal Cys-tag and a C-terminal Avi-tag were genetically added to the proteins for site-directed immobilization. Despite only minor differences regarding the number, positioning, and sequence context of lysine residues within the different RBD variants, those differences led to a dramatic change in their functionality after EDC/NHS coupling. In contrast, site-specific biotinylation of the proteins alongside targeted immobilization on streptavidin- or neutravidin-modified liposomes resulted in restored functionality and enhanced storage stability across all variants. The developed adaptable liposome-based test showed excellent correlation with an established pseudovirus neutralization test and could identify variations in neutralization patterns of Alpha/Delta and Omicron variants in patient sera. The study highlights the benefits of using neutravidin-liposomes for site-directed protein immobilization with independence from the proteins’ amino acid sequences, enhanced storage stability, and applicability to various biotinylation strategies, serving as a versatile platform technology that can also be applied to the coupling of other proteins or peptides used for diagnostic purposes.