OBJECTIVES: There is a crucial unmet need for biomarker-guided diagnostic and prognostic enrichment in clinical trials evaluating immune modulating therapies in critically ill patients. Low monocyte expression of human leukocyte antigen-DR (mHLA-DR), considered as a reference surrogate to identify immunosuppressed patients, has been proposed for patient stratification in immunostimulation approaches. However, its widespread use in clinic has been somewhat hampered by technical constraints inherent to flow cytometry technology. The objective of the present study was to evaluate the ability of a prototype multiplex polymerase chain reaction tool (immune profiling panel [IPP]) to identify immunosuppressed ICU patients characterized by a low mHLA-DR expression. DESIGN: Retrospective observational cohort study. SETTING: Adult ICU in a University Hospital, Lyon, France. PATIENTS: Critically ill patients with various etiologies enrolled in the REAnimation Low Immune Status Marker study (NCT02638779). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: mHLA-DR and IPP data were obtained from 1,731 blood samples collected from critically ill patients with various etiologies and healthy volunteers. A partial least square regression model combining the expression levels of IPP markers was trained and used for the identification of samples from patients presenting with evidence of immunosuppression, defined here as mHLADR less than 8,000 antibodies bound per cell (AB/C). The IPP gene set had an area under the receiver operating characteristic curve (AUC) of 0.86 (95% CI 0.83–0.89) for the identification of immunosuppressed patients. In addition, when applied to the 123 patients still in the ICU at days 5–7 after admission, IPP similarly enriched the number of patients with ICU-acquired infections in the immunosuppressed group (26%), in comparison with low mHLA-DR (22%). CONCLUSIONS: This study reports on the potential of the IPP gene set to identify ICU patients presenting with mHLA-DR less than 8,000 AB/C. Upon further optimization and validation, this molecular tool may help in the stratification of patients that could benefit from immunostimulation in the context of personalized medicine.