The response against tissue injury and infection begins with the early activation of molecular and cellular elements of the inflammatory and immune response. Severe tissue injury, necrosis, and infection induce imbalanced inflammation associated with leukocyte over-stimulation and excessive or dysregulated release of cellular mediators. Clinical and experimental studies have shown that these mediators are directly related to progressive post-injury complications. Persistent increased levels of pro-inflammatory mediators produce tissue injury. Excessive production and activity of anti-inflammatory mediators cause anergy and/or immune dysfunction with increased susceptibility to infection. Leukocyte activation is assessed by cell surface phenotype expression, cellular mediators determination, or by measuring functional responses using isolated cells. Potential routine clinical uses are: evaluation of severity and prognosis in critically ill patients, immunomonitoring of sepsis, and detection of tissue injury, necrosis, and infection. In practice, the determination of cellular activation markers is restricted by a limited number of automated methods and by the cost of reagents. The availability of flow cytometry and immunoassay automated systems can contribute to a wider use in practice. Here we review the immunopathophysiology of polymorphonuclear neutrophil, monocyte, macrophage, and lymphocyte activation in response to tissue injury and infection. In addition, laboratory methods for their determination, and clinical applications in practice, are discussed.