Advanced glycation end products (AGEs) are formed through the reaction/modification of proteins by saccharides (e.g., glucose and fructose) and their intermediate/non-enzymatic products [e.g., methylglyoxal and glyceraldehyde (GA)]. In 2017, Dr. Takanobu Takata et al. developed the novel slot blot method to quantify intracellular GA-derived AGEs (GA-AGEs). Although the original method required nitrocellulose membranes, we hypothesized that the modified proteins contained in the AGEs may be effectively probed on polyvinylidene difluoride (PVDF) membranes. Because commercial lysis buffers are unsuitable for this purpose, Dr. Takata developed the slot blot method using an in-house-prepared lysis buffer containing 2-amino-2-hydromethyl-1,3-propanediol (Tris), urea, thiourea, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) that effectively probes AGEs onto PVDF membranes. The slot blot method also entails the calculation of Tris, urea, thiourea, and CHAPS concentrations, as well as protein and mass to be probed onto the PVDF membranes. GA-AGE-modified bovine serum albumin (BSA, GA-AGEs-BSA) is used to draw a standard curve and perform neutralization against a non-specific combination of anti-GA-AGEs antibodies, thereby enabling the quantification of GA-AGEs in cell lysates. This paper presents the detailed protocol for slot blot analysis of intracellular GA-AGE levels in C2C12 cells.