Abstract Background: Mass spectrometry (MS)-based 17-hydroxyprogesterone (17OHP) methods show considerable variation in results in external quality assurance (EQA) programs. An understanding of the current status of MS-based serum/plasma 17OHP quantification is important to facilitate harmonization. Methods: A 50-item e-survey related to (1) laboratory characteristics, (2) pre-analytical considerations and (3) analysis of 17OHP was developed and circulated to clinical MS laboratories via professional associations in Asia Pacific, Europe and North America. Results: Forty-four laboratories from 17 countries completed the survey. Sample preparation varied between laboratories with protein precipitation and liquid-liquid extraction being the most common processes. Analyte separation was most commonly achieved by liquid chromatography (LC) using a C18 column and mobile phases of water, methanol and formic acid. The ions selected for quantification were 331>97 m/z or 331>109 m/z. Alternative transition ions were used as qualifiers. Twenty-seven of 44 respondents reported preparing their calibrators in-house and variations in material purity and matrix were evident. Nine of 44 laboratories did not participate in an EQA program, and half did not know if their method separated out isobars. The reference intervals, and also their partitioning, reported by the laboratories were highly discrepant, in some cases, by multiple folds. Conclusions: Although MS-based methods are similar in many facets, they are highly disparate. Five recommendations have been developed as an outcome of this survey to support the continued improvement of analysis of serum/plasma 17OHP by MS.