The use of polyethylene glycol 400 (PEG 400) as an excipient in oral formulations can have profound and differing effects on drug bioavailability in men and women; therefore an understanding of the pharmacokinetics of this excipient is required. A direct injection electrospray selected ion monitoring mass spectrometry methodology was developed and validated for the quantitation of PEG 400 excreted in human urine after oral administration. The most abundant ions corresponding to PEG 400 oligomers at m/z 365, 409, 453, 497, 541, and 585 were used for selected ion monitoring (SIM). Pre-dose urine of volunteers was spiked with various amounts of PEG 400 to generate calibration curves over the concentration range 2.5-90 μg/mL for all SIM channels. The relative standard deviations of intra- and inter-day analysis of PEG 400 in human urine were lower than 11.8% and bias percentage was less than 9.7%. This specific method for relative quantitation of PEG 400 was then used to analyse urine samples with minimal sample preparation. Urine samples of twelve healthy volunteers (six men and six women) who received 0.75 g and 1.5 g PEG 400 on two separate occasions were collected over 24h. On average 36.5% of the orally administered dose of PEG 400 was recovered in the urine of the volunteers, with no significant difference observed between men and women.