Daily rhythms of behaviors and physiologies are driven by transcriptional-translational negative feedback loops of clock genes and encoded clock proteins (Bass and Takahashi Science 330:1349-1354, 2010; Brown et al. Dev Cell 22:477-487, 2012). Posttranslational modifications of clock proteins, including protein phosphorylation, play an essential role for normal oscillation of the circadian clock through regulation of their activities, stabilities, interactions, and intracellular localization (Gallego and Virshup Nat Rev Mol Cell Biol 8:139-148, 2007; Hirano et al. Nat Struct Mol Biol 23:1053-1060, 2016). In this chapter, we describe detailed methods for quantitative analysis of phosphorylation levels of clock proteins, particularly focusing on circadian phosphorylation of CLOCK, BMAL1, and their complex (Yoshitane et al. Mol Cell Biol 29:3675-3686, 2009).