Cellular senescence, which can be defined as a stress response preventing the propagation of cells that have accumulated potentially oncogenic alterations, is invariably associated with a permanent cell cycle arrest. Such an irreversible blockage is mainly mediated by the persistent upregulation of one or more cyclin-dependent kinase inhibitors (CKIs), including (though not limited to) p16( INK4A ) and p21( CIP1 ) and p27( KIP1 ). CKIs operate by binding to cyclin-dependent kinases (CDKs), de facto inhibiting their enzymatic activity. Here, we provide an immunoblotting-based method for the detection and quantification of CKIs in vitro and ex vivo, together with a set of guidelines for the interpretation of results.