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Herein, we report the development of a NanoBRET\u2010based assay platform based on the fluorescent ligand LT221 (<jats:bold>5<\/jats:bold>), to detect intracellular binding to CCR6 and CXCR1, two chemokine receptors that have been pursued as promising drug targets in inflammation and immuno\u2010oncology. Our assay platform enables cell\u2010free as well as cellular NanoBRET\u2010based binding studies in a nonisotopic and straightforward manner. By combining this screening platform with a previously reported CXCR2 assay, we investigated CXCR1\/CXCR2\/CCR6 selectivity profiles for both known and novel squaramide analogues derived from navarixin, a known intracellular CXCR1\/CXCR2 antagonist and phase\u2005II clinical candidate for the treatment of pulmonary diseases. By means of these studies we identified compound <jats:bold>10<\/jats:bold>, a previously reported <jats:italic>tert<\/jats:italic>\u2010butyl analogue of navarixin, as a low nanomolar intracellular CCR6 antagonist. 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