Among the regulatory mechanisms that control gene expression, mRNA translation plays a fundamental role in determining the functional output of the transcriptome in cells. This is particularly relevant in immune cells, where rapid and tightly regulated translation is essential for maintaining immune homeostasis. Analyzing the translational status of transcripts, as well as the mechanisms underlying their regulation, can provide valuable insights into the key factors influencing physiological and pathological phenotypes. Here, we describe our routinely used protocol for polysome profiling, a method that enables the analysis of mRNA translation at both the individual transcript and genome-wide levels.