A high-throughput method combining liquid handling system and 96-well microplate pipetting format was developed for total sugar determination. With this new method, we characterized diverse sugar accumulation in sorghum varieties. Sweet sorghum accumulates large amounts of sucrose in its stalk and, therefore, has emerged as one important bioenergy crop. The commonly used sugar measurement, Brix, limits the characterization of internode variation of the sugar concentrations due to its low throughput. Here we developed a low-cost, high-throughput method to determine profiles of total sugars in sorghum internodes with a liquid handling system-based sample preparation and a phenol-sulfuric acid assay in 96-well microplate format. The present method generates results highly correlated with commonly used Brix measurements (r = 0.922). The inter-assay coefficient of variation ranged from 4.8 to 7.6%. The present method can reliably estimate mixed sugars composed of 80% sucrose. We characterized the profiles of 35 sorghum accessions and identified 21 accessions with significantly different sugar concentrations between internodes either due to dried-up internodes or concentration differences. As a high-throughput alternative to Brix measurements, the new method makes it possible to phenotype total sugars from large numbers of internode samples and, therefore, will be useful for genetic and breeding purposes.