The enzyme-linked immunospot assay (ELISPOT) is a technique widely used to enumerate the number of immune cells secreting a specific protein, such as antibodies or cytokines. A limitation with the ELISPOT assay is that it can only be used to detect a single protein of interest. Recently, the ELISPOT technique has been modified to use fluorophores allowing multiple secreted proteins to be detected simultaneously. This technique has greatly enhanced the ability to identify cells secreting multiple proteins, but has not been used to its fullest potential. We wished to accurately quantify the expression of antigen-specific antibody from a single plasma cell and to determine whether plasma cells recovered from different locations had different secretion rates. To achieve this we analyzed fluorospot images quantitatively using Mira MX 7 UL Astronomy software, and coupled this data with a quantitative ELISA to determine secretion rates from individual cells. Using this technique we were able to determine that plasma cells recovered from the peripheral blood secreted the most antibody (1.667 ng/cell/12 h) while splenic antibody secreting cells the least (0.399 ng/cell/12 h). We were able to quantify a 150 fold difference in antibody secretion between cells, with most plasma cells divided into two groups, low secretors (<0.1 ng/cell) or high secretors (>2 ng/cell). We believe this technique will be particularly useful for examining the secretion ratio of two proteins secreted from an individual cell, allowing us to determine if secretion is fixed or variable.