Nuclear magnetic resonance (NMR)-based metabolic profiling of biofluids and tissues are of key interest to enhance biomarker discovery for disease, drug efficacy and toxicity studies. Urine and blood plasma/ serum are the biofluids of most interest as they are the most accessible in both clinical and preclinical studies. However, proteinaceous fluids, such as blood serum or plasma, represent the greatest technical challenge since the chemical shift (d) and line-width (m 1/2 ) of internal standards currently used for aqueous NMR samples are greatly affected by protein binding. We have therefore investigated the suitability of 4,4-dimethyl-4-silapentane-1-ammonium trifluoroacetate (DSA) as a universal internal standard for biofluids. Proton ( 1 H) NMR spectroscopy was used to determine the effect of serum pH (3, 7.4 and 10) and DSA concentration on the overall lineshape and position of the trimethylsilyl resonance of DSA. The results were compared to that of 3-(trimethylsilyl)propionic acid sodium salt (TSP). Both the chemical shift and line-width of the DSA peak were not significantly affected by pH or DSA concentration, whereas these parameters for TSP showed large variations due to protein binding. Furthermore, the peak area of DSA correlated linearly with its concentration under all pH conditions, whilst no linear correlation was observed with TSP. Overall, in contrast to TSP, these results support the use of DSA as an accurate universal internal chemical shift reference and concentration/normalisation standard for biofluids. In the case of proteinaceous biofluids such as serum, where no current standard is available, this offers a considerable saving in both operator and spectrometer time.