Background: Over the past decade the use of LC-MS/MS has increased significantly in the hospital laboratories. Clinical laboratories have switched from immunoassays to LC-MS/MS methods due to the... Show moreBackground: Over the past decade the use of LC-MS/MS has increased significantly in the hospital laboratories. Clinical laboratories have switched from immunoassays to LC-MS/MS methods due to the promise of improve-ments in sensitivity and specificity, better standardization with often non-commutable international standards, and better between-laboratory comparison. However, it remains unclear whether routine performance of the LC-MS/MS methods have met these expectations. Method: This study examined the EQAS results, from the Dutch SKML, of serum cortisol, testosterone, 25OH-vita-minD and cortisol in urine and saliva over 9 surveys (2020 to first half of 2021). Results: The study found a significant increase in the number of compounds and in the number of results measured in the different matrices, with LC-MS/MS over a period of eleven years. In 2021, approximately 4000 LC-MS/MS results were submitted (serum: urine: saliva = 58:31:11%) compared to only 34 in 2010. When compared to the individual immunoassays, the LC-MS/MS based methods for serum cortisol, testosterone and 25OH-vitaminD showed comparable but also higher between-laboratory CVs in different samples of the surveys. For cortisol, testosterone and 25OH-vitaminD the median CV was 6.8%, 6.1% and 4.7% respectively for the LC-MS/MS compared to 3.9-8.0%,4.5-6.7%, and 7.5-18.3% for immunoassays. However, the bias and imprecision of the LC-MS/MS was better than that of the immunoassays. Conclusion: Despite the expectation that LC-MS/MS methods would result in smaller between-laboratory dif-ferences, as they are relatively matrix independent and better to standardize, the results of the SKML round robins do not reflect this for some analytes and may be in part explained by the fact that in most cases laboratory developed tests were used. Show less