Glycogen, a soluble multi-branched glucose homopolysaccharide, is composed of chains of α-1,4-linked glucose residues interconnected by α-1,6-linked branches. The classical biosynthetic pathway involves phosphoglucomutase (Pgm), glucose-1-phosphate adenylyltransferase (GlgC or GlgCD), glycogen synthase (GlgA) and branching enzyme (GlgB). Phosphoglucomutase converts glucose-6-phosphate into glucose-1-phosphate, which serves as a substrate for ADP-glucose synthesis catalyzed by GlgC or GlgCD. Then, GlgA catalyzes the transfer of glucosyl units from ADP-glucose to the elongating chain of linear α-1,4-glucan. GlgB subsequently cleaves off portions of the glucan and links it to internal glucose molecules in existing chains via α-1,6 glycosidic bonds to form the glycogen structure. Glycogen breakdown is mediated by glycogen phosphorylase (GlgP) and debranching enzyme (GlgX), which catalyze the sequential phosphorolysis of α-1,4-glucosyl linkages in the glucan chain from the non-reducing ends and debranching of the limit dextrins generated by GlgP, respectively. An increasing number of studies have revealed the involvement of glycogen metabolism in a multitude of physiological functions in some prokaryotes beyond the function of synthesizing energy storage compounds. Lactobacillus acidophilus NCFM was the first probiotic lactic acid bacterium demonstrated to possess a functional glycogen biosynthesis pathway that is involved in its growth, bile tolerance and complex carbohydrate metabolism (Goh and Klaenhammer, 2013). The following qualitative (for detection of intracellular glycogen) and quantitative (for measurement of intracellular glycogen content) intracellular glycogen assay protocols for Lactobacillus acidophilus (L. acidophilus) were modified from previous works (Govons et al., 1969; Law et al., 1995; Parrou and Francois, 1997) and should be applicable to other lactic acid bacteria as well as most microorganisms.