The Griess assay is used to measure nitric oxide concentrations in liquid solutions after reaction into nitrite. The assay is challenging when applied to cell culture supernatants. During optimization, we focused on the anti-inflammatory potential of test compounds in murine RAW264.7 macrophages. This led to (i) the required inductivity of cells by lipopolysaccharide (LPS) and allowed (ii) the characterization of putative anti-inflammatory test compounds with high sensitivity. The modifications reported here prominently improved resolution and efficiency of the widely used Griess assay and are of broad interest for studies on the pharmacological modulation of macrophages activation. Copyright © 2017 Elsevier B.V. All rights reserved.