Abstract Objectives Our recent survey of 44 mass spectrometry laboratories across 17 countries identified variation in internal standard (IS) choice for the measurement of serum/plasma 17α-hydroxyprogesterone (17OHP) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The choice of IS may contribute to inter-method variations. This study evaluated the effect of two common isotopically labeled IS on the quantification of 17OHP by LC-MS/MS. Methods Three collaborating LC-MS/MS laboratories from Asia, Europe and Australia, who routinely measure serum 17OHP, compared two IS, (1) IsoSciences carbon-13 labeled 17OHP-[2,3,4-¹³C₃], and (2) IsoSciences deuterated 17OHP-[2,2,4,6,6,21,21,21-²H]. This was performed as part of their routine patient runs using their respective laboratory standard operating procedure. Results The three laboratories measured 99, 89, 95 independent samples, respectively (up to 100 nmol/L) using the ¹³C- and ²H-labeled IS. The slopes of the Passing-Bablok regression ranged 0.98–1.00 (all 95% confidence interval [CI] estimates included the line of identity), and intercept of <0.1 nmol/L. Average percentage differences of −0.04% to −5.4% were observed between the two IS materials, which were less than the optimal bias specification of 7% determined by biological variation, indicating no clinically significant difference. The results of 12 Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) proficiency samples (1–40 nmol/L) measured by the laboratories were all within the RCPAQAP analytical performance specifications for both IS. Conclusions Overall, the comparison between the results of ¹³C- and ²H-labeled IS for 17OHP showed good agreement, and show no clinically significant bias when incorporated into the LC-MS/MS methods employed in the collaborating laboratories.