Batch variation in Tween 60 has shown to influence the rheological properties of semisolid emulsions. MS (LC-MS, GC-MS, MS(n)) and NMR ((13)C, (1)H, (1)H COSY and HMBC) techniques were used to analyze and compare the composition of two batches of Tween 60 with particular emphasis on the number of POE groups and their distribution within the molecule. Acid and saponification values were also determined. The batches contained different proportions of components (sorbitan polyethoxylates, sorbitan monoester-diester-polyethoxylates and isosorbide monoester-diester-polyethoxylates). The number of POE groups were averaged over the four sites in sorbitan and the two sites in isosorbide molecules. The batches differed from each other in terms of (i) the POE sorbitan stearate/POE sorbitan palmitate ratios (batch 1, 3:2 and batch 2, 4:5), (ii) the ratio of sorbitans to isosorbides (batch 1, 2:3; batch 2, 7:13); and (iii) the acid values (batch 1, 3.1; batch 2, 0). It is concluded that liquid chromatography combined with electrospray mass spectrometry and ion trap separation is a useful tool for establishing the compositional profile of different batches of Tweens. (1)H NMR could provide a simple and rapid pharmacopoeial test for the ratio of sorbitan to isosorbide in Tweens.