Current multiplex assay technologies on the market for soluble targets are frequently associated with high reagent and instrument cost, inconsistent assay performance, inaccurate data analysis, and lack of good reproducibility.  To address the issues, we developed four multiplexed bead-based assay panels for simultaneous quantification of soluble markers using standard flow cytometers available in many laboratories. Our human and mouse T help (Th) panels allow quantification of 13 cytokines, including interleukins (IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22), IFN-g and TNF-α, which are collectively secreted by Th1, Th2, Th9, Th17, Th22 and T follicular cells.   Our human and mouse proinflammatory chemokine panels detect 13 chemokines, including MCP-1 (CCL2), RANTES (CCL5), IP-10 (CXCL10), Eotaxin (CCL11) , TARC (CCL17), MIP-1α (CCL3), MIP-1β (CCL4), MIG (CXCL9), and MIP-3α (CCL20). The human chemokine panel also includes ENA-78 (CXCL5), GROα (CXCL1), I-TAC (CXCL11), and IL-8 (CXCL8), and the mouse chemokine panel contains LIX (CXCL5), KC (CXCL1), BLC (CXCL13) and MDC (CCL22). Each antibody pair was carefully selected for assay specificity, sensitivity, accuracy and reproducibility. The assay panels have been validated in appropriate biological samples, detecting changes as expected.  Compared with similar assay products on the market, LEGENDplex™ assays provide equal or better analytical performance with reduced cost, better performance consistency, and greater flexibility. To facilitate analysis of data (FCS 2.0, 3.0, 3.1, and list mode files) from different flow cytometers, a software package with an intuitive interface, accurate curve-fitting, and robust reporting was developed and is offered free of charge. The assays and software can be used for quantitative and qualitative analysis of soluble biomarkers from serum, plasma, cell culture supernatant, and other sample types, offering a more economical alternative for multiplex solutions.