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Macrophages are phagocytic cells from the innate immune system that are critical for tissue homeostasis and form the first line of host defense against invading pathogens. The zebrafish larva is an exquisite model to decipher the transcriptional response of macrophages after injury. We used a macrophage reporter line in which an mfap4 promoter drives the expression of a farnesylated mCherry fluorescent protein to label macrophages and we performed tissue dissociation, cell isolation by Fluorescence Activated Cell sorting and RNA preparation. The two bottlenecks are (i) the dissociation of the embryos that often relies on cell suspension steps that alter the activation status of immune cells, and (ii) obtaining high RNA integrity for gene expression analysis from a small number of isolated macrophages. Here, we describe (i) the dissociation of cells from whole Tg(mfap4:mCherry-F) zebrafish larvae using an enzyme-free and osmotically controlled buffer, (ii) the sorting of fluorescent macrophages by FACS and (iii) the preparation of high quality RNAs for meaningful gene expression analysis from a small number of isolated macrophages. • An optimized protocol in 5 steps to extract high quality RNAs from zebrafish macrophages. • A cell dissociation method using an enzyme-free and osmotically controlled buffer to prevent the alteration of macrophage activation status and limit cell mortality. • Production of high integrity RNAs from a small number of isolated macrophages.