The chemical analysis of hop-derived humulone and isohumulone components in hops and beer was investigated using multiple injections in a single experimental run (MISER) extracted ion LC-MS. Comparative analysis of the humulone and lupulone contents of 12 hop varieties was accomplished in a 12 min run, an equivalent time of 1 min per hop sample. Rapid analysis of a group of 70 different beers in 74 min provided a comparative assessment of the amount of humulones (α-acids) and the isomerized isohumulones (iso-α-acids) present, also in an equivalent time of 1 min per sample. The relative humulone/isohumulone content was evaluated in a scatter plot, which allowed profiling of various craft and commercial beer styles. The MISER LC-MS approach allows for high-throughput analysis and a significant time savings. Together, these provide opportunities for increasing analysis sampling in production or a simple, rapid chemical analysis to accompany educational or classroom instruction.