The undecapeptide substance P (SP) is a member of the tachykinin family of neurotransmitters, which has a pivotal role in the regulation of inflammatory and immune responses. One of the major barriers to the study of the in vivo role of SP in a number of immune disorders is the accurate measurement of SP in fluids. This is reflected in the variability of reported SP levels in serum and plasma of humans in both healthy and diseased states. This study was initiated in order to identify sources of variability by the comparative evaluation of the influences of sample preparation and analytical detection methods on the measurement of SP in plasma. The results indicate that sample preparation (peptide extraction versus no extraction) and the choice of analytical method for SP quantitation may yield significantly different values and may contribute to the variability in SP values reported in the literature. These results further emphasize the need for careful consideration in the selection of methods for SP quantitation, as well as caution in the interpretation and comparison of data reported in the literature.