Liquid chromatography tandem mass spectrometry (LCMS/MS) methods are considered superior to immunoassay for the analysis of serum 25-hydroxy vitamin D (25-OHD) due to improved performance and potential cost benefits. As LCMS/MS is appropriate for the analysis of other clinically relevant hormones tandem mass spectrometers are becoming commonplace in UK clinical laboratories. However, like immunoassay, LCMS/MS methods are not foolproof and inappropriate use of LCMS/MS methods can have an adverse impact on patient care. Triple quadrupole mass spectrometers allow the monitoring of several fragment ions derived from a single molecular ion. This allows quantitation using one fragment (the quantifier) and analysis of other fragments (qualifiers) to confirm the identity of the molecular ion. Examples are provided as to the use of qualifier ions to detect both endogenous and exogenous interferences that would otherwise bias LCMS/MS 25-OHD methods. During optimisation of an LCMS/MS 25-OHD method, the use of qualifier ions detected an endemic interference which was significant in 1023 of the 3060 patient samples studied using a commonly used quantifier ion of m/z 211. We identified three cases that were classified as severely 25-OHD deficient (<12.5 nmol/l) using a 383257 transition who would be classified as vitamin D replete (>50 nmol/l) using the 383211 transition. The interference was still present despite use of a variety of sample preparation methods (liquid/liquid extraction, off-line and on-line solid phase extraction). Accurate mass MS studies show the interference to have a mono-isotopic m/z of 383.2964 compared to m/z 383.3308 for 25-OHD. Further studies suggest that this is an exogenous compound, probably exuded from the sample collection tubes. The isobaric C-3 epimer of 25-OHD [3-epi-25(OH)D3] has also been shown to be a potential interference in LCMS/MS based serum 25-OHD assays. The use of appropriate qualifier ions to rule out the presence of 3-epi-25(OH)D3 is demonstrated.