Remarkable progress in basic, translational, and clinical cancer research has been observed during the last decade. This has opened possibilities for the development of novel diagnostics and therapeutic approaches and created opportunities for personalized medicine. Cancer biomarkersBiomarkers are key players in human cancer progression, both peripheral and at the site of tumor. Through reliable techniques detecting biomarkersBiomarkers, cancer can thus be predicted, diagnosed and progression and response to therapy can be followed. MultiplexMultiplex analysis of biomarkersBiomarkers in small blood volumes allows for rapid quantificationQuantification of large number of circulating analytes. The LuminexLuminex technique allows multiple biomarkersBiomarkers to be measured simultaneously in small volumes and provides a convenient and sensitive tool for the detection of large number of extracellular secreted biomarkersBiomarkers to be used in prediction and therapy prognosis in cancer. The technique is based on so-called microspheres (beads) that serve as a solid phase for molecular detection. These individually dyed microbeads have monoclonal antibodies directed against the biomarkerBiomarkers of interest and allow simultaneous detection of up to hundreds of biomarkersBiomarkers in a dual-laser flow analyzer. BiomarkersBiomarkers can be detected in serum- and plasmaPlasma samples as well as in cell culture supernatants from in vitro cultured and stimulated cells, e.g., peripheral blood mononuclear cells (PBMC)Peripheral blood mononuclear cells (PBMC) or cancer cell lines. The need for robust detection of biomarkersBiomarkers for prediction as well as outcome of cancer therapy progression is of great importance. This chapter describes the LuminexLuminex technique for detection of biomarkersBiomarkers associated with cancer by magnetic beadMagnetic beads sandwich immunoassayImmunoassay, with focus on some important pre-analytic factors, e.g., cell separation and cryopreservation and thawing of PBMCPeripheral blood mononuclear cell (PBMC) that may affect the outcome of detection of biomarkersBiomarkers. The LuminexLuminex technique is thus one way to discover biomarkersBiomarkers to predict, prognose, and improve clinical outcome of cancer.