Sequential chemical depletion of serum coupled to C18 sequential extraction of peptides as a rapid tool for human serum multiple profiling is herein presented. The methodology comprises depletion with DTT and then with ACN; the extract thus obtained is then summited to fast protein digestion using ultrasonic energy. The pool of peptides is subsequently concentrated using C18-based Zip-tips and the peptides are sequentially extracted using different concentrations of ACN. Each extract is mass-spectrometry profiled with MALDI. The different spectra thus obtained are then successfully used for classification purposes. A total of 40 people, comprising 20 healthy and 20 non-healthy donors, were successfully classified using this method, with an excellent q-value<0.05. The proposed method is cheap as it entails few chemicals, DTT and ACN, simple in terms of handling, and fast. In addition, the methodology is of broad application as it can be used for any study applied to serum samples or other complex biological fluids.