Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition. Primary HLH occurs early in life due to monogenic biallelic mutations affecting lymphocyte cytotoxicity. Secondary HLH occurs mostly in adults secondary to infections, lymphoma or rheumatic diseases. In this latter setting, lymphocyte cytotoxicity status is not known. We conducted a systematic evaluation of NK cell cytotoxicity in adult patients with secondary HLH. Adult patients with secondary HLH were prospectively studied ex vivo for total lymphocyte count and subtypes, NK cell phenotype, perforin expression and degranulation, natural or ADCC cytotoxicity, in comparison with patients affected by the same underlying diseases without HLH (disease controls, DC) and with healthy controls (HC). Screening for variants of cytotoxity genes was systematically performed. 68 patients were included in the HLH group, 34 in the DC and HC groups, respectively. In HLH patients, a severe and transient lymphopenia, an activated NK cell phenotype (increased CD69, ICAM-1, HLADR, CCR5 expression) and a decreased capacity of interferon gamma production were observed. Mean perforin expression was normal, degranulation tests and NK cell cytotoxicity were not different from DC. A monoallelic variant of uncertain significance affecting one of the lymphocyte cytotoxicity genes, or the perforin variant A91V, was observed in almost 50% of the patients. We detected no major intrinsic cytotoxicity dysfunction in secondary HLH compared to DC and no predicted pathogenic gene variant. The activated NK phenotype profile associated with decreased interferon gamma production appear similar to other hyperinflammatory diseases such as sepsis or systemic juvenile idiopathic arthritis.