Rapid multiplex cell surface marker analysis can expedite investigations in which large number of antigens need to be analyzed. Simultaneous analysis of multiple surface antigens at the same level of sensitivity is however limited in the current golden standard analysis method, flow cytometry. In this paper we introduce a surface plasmon resonance imaging (SPRi)-based technique for 44-plex parameter analysis using a single sample, in less than 20 min. We analyzed the expression on cells from five different cancer cell lines by SPRi on a 44-plex antibody array including 4 negative controls and compared the output with flow cytometry. The combined correlation of the markers that showed expression by flow cytometry was 0.76. The results demonstrate as a proof of principle that SPRi can be applied for rapid semi-quantitative multiplex cell surface marker analysis.