Methods for preparation of cells for analysis of biomarkers are disclosed. In one aspect, the method for preparation of cells for analysis of biomarkers includes contacting a sample that contains a population of cells with at least one modulating substance at a first temperature, thereby producing a modulated cell population; contacting the modulated cell population with at least one antibody that is directed to a cell surface biomarker at a second temperature that is lower than the first temperature, thereby producing an extracellularly stained cell population; and contacting the extracellularly stained cell population with one or more reagents that fixes and permeabilizes the cells, thereby producing a fixed and permeabilized cell population.