Homogalacturonans, the most abundant pectins of the plant cell wall, can be methylesterified at the C-6 position of the galacturonic acid residues. Demethylesterification of cell wall pectins is catalyzed by apoplastic pectin methylesterases (PMEs). Several plant developmental processes and plant-environment interactions involve PME-mediated cell wall modification, as it promotes the formation of Ca²⁺cross-links along the stretches of the demethylesterified galacturonic acid residues (Wolf et al., 2009; Müller et al., 2013), and thus influences the biophysical properties of plant cell walls. Here, we describe a protocol that can be used to estimate the activity of PMEs in a total soluble protein extract from plant or seed tissues. Soluble protein is extracted from the plant/seed materials, and a coupled enzyme assay is performed, according to a procedure modified from Grsic-Rausch and Rausch (2004). The methanol released from methylesterified pectins as a result of PME activity is oxidized to formaldehyde by alcohol oxidase. The formaldehyde is then used as an electron donor by formaldehyde dehydrogenase to reduce NAD+ to NADH. The formation of NADH from NAD+ is followed spectrophotometrically, and used to estimate the PME activity in the protein extract.