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The potential of dried blood, plasma and urine spots, deposited on a paper substrate, combined with reversed-phase ultra performance liquid chromatography and orthogonal acceleration time-of-flight mass spectrometry (UPLC-oaToFMS), with electrospray ionization (ESI), has been investigated for global metabolic profiling. When compared to analysis using protein precipitated plasma, both blood and plasma spots gave comparable profiles and numbers of ions etc., using both positive and negative ESI. In the case of urine the results for spots obtained in positive ESI were also comparable to those of the untreated sample, but with negative ESI, a significant reduction in ions was noted for spotted samples. These preliminary data suggest that, with further optimization, biofluid spotting may provide an alternative to conventional methods for this type of work in suitable applications.