Mitosis is characterized by the activation of mitotic kinases and inhibition of phosphoprotein phosphatases (PPPs), resulting in a stark increase in substrate phosphorylation that initiates mitotic progression. This balance of activities is reversed at the metaphase-to-anaphase transition. Besides the antagonistic action of protein kinases and phosphatases at the level of substrate phosphorylation, it is becoming apparent that reciprocal regulation of PPPs by protein kinases and vice-a-versa is an essential regulatory mechanism crucial for the generation of distinct cell cycle phases. While kinases have been extensively studied in this regard, we are just starting to elucidate PPP regulation. PPPs are responsible for the majority of serine/threonine dephosphorylation in eukaryotic cells and achieve substrate selectivity and specificity through the formation of holoenzyme complexes. Thus, the identification and quantification of PPP holoenzyme complexes in different cell cycle phases is important to elucidate their regulatory function. We have recently developed a mass spectrometry-based chemical proteomics approach for the enrichment, identification, and quantification of endogenous PPP holoenzyme complexes. Here, we combine PPP and kinase profiling to investigate the phosphorylation-dependent regulation of PPPs in mitosis. Comparison with global protein abundances is employed to determine the regulatory mechanism governing these changes. We demonstrate the utility of this approach by identifying that Cyclin-dependent kinase 1 (Cdk1) as the kinase that phosphorylates the catalytic subunit of PP2A (PP2Ac) on threonine 304. We show that this phosphorylation disrupts PP2A-B55 holoenzyme formation and thereby regulates mitotic entry and exit through alteration of substrate phosphorylation.