Objectives: In response to increasing numbers of states in the US that test newborn babies for cystic fibrosis (CF), the Newborn Screening Quality Assurance Programme initiated a pilot proficiency testing programme for immunoreactive trypsinogen (IRT), the biomarker for CF. Dried blood spot specimens (DBS) were used to evaluate the performance of laboratories that screen babies for CF. Methods: DBS were prepared from human whole blood enriched with physiologically relevant levels of IRT. Various methods of making IRT-enriched DBS were used to optimize the recovery and stability of the biomarker, including preparation of DBS from either intact or lysed red blood cells, varying the timing of IRT addition to blood before dispensing onto filter paper, adding a protease inhibitor cocktail, and treating serum with charcoal before IRT enrichment. The recovery and stability of IRT in DBS were assessed. Newborn screening laboratories were offered the opportunity to test blind-coded DBS in the pilot PT programme. Results: IRT was stable in the filter paper matrix when stored for one year at either −20°C or 4°C. Fifty percent more IRT was recovered from DBS prepared with lysed red blood cells where the IRT was added to blood just before dispensing; however, protease inhibitors did not improve IRT recovery. Conclusions: IRT in the DBS matrix is stable and can be shipped worldwide under ambient conditions. Optimal IRT recovery was achieved by adjustment of DBS production practices. Laboratories receiving specimens accurately measured IRT by a variety of commercial and in-house methods.