The invention provides methods of preparing a test sample for use in an assay for detecting an analyte bound by an intracellular ligand. The methods typically involve contacting the test sample with an assay reagent comprising: a lysis reagent; and a protease that has proteolytic activity for said intracellular ligand; to form a mixture compatible for use in an immunoassay without subsequent extraction steps. Other aspects of the invention include related immunoassays and test kits.