Cancer immunotherapy based on immune checkpoint blockade (ICB) has demonstrated durable clinical benefit in patients with immunogenic tumors, representing a new alternative in the metastatic scenario. However, innate and acquired resistance is the Achilles heel of this revolutionary therapeutic approach, together with the occurrence of mostly autoimmune adverse effects, particularly in the most effective regimen, the combination immunotherapy. This, together with the high treatment cost, has triggered an important crusade to identify biomarkers that can be used to detect ICB responders before and during treatment that could be used for further clinical stratification and monitoring. Because ICB implementation is relatively recent, the initial works in the field of mechanisms and biomarkers of resistance included very modest cohorts; however, that limitation led to a maximized exploitation of the clinical material, which yielded biomarkers on most OMICS, from genomics to microbiome or radiogenomics. One of the most critical areas of current immunotherapy research is the urgent finding of specific novel predictive biomarkers that could identify individuals who would benefit from ICB. The objective of this chapter is to provide an overview of the current knowledge on checkpoint biology by reviewing the different emerging therapeutically relevant immune checkpoint blockade response biomarkers and their modulation of immune checkpoint signaling at multiple levels.