Background In cystic fibrosis newborn screening (CFNBS), immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP) can be used as screening parameters. We evaluated the IRT × PAP product as second-tier parameter in CFNBS in newborns with elevated IRT. Methods Data on 410,111 screened newborns including 78 patients with classical cystic fibrosis (CF) from two European centers were retrospectively analyzed by discrimination analysis to identify a screening protocol with optimal cutoffs. We also studied differences in PAP measurement methods and the association of IRT and PAP with age. Results PAP values differed systematically between fluorometric and photometric assays. The IRT × PAP product showed better discrimination for classical CF than PAP only as second-tier screening parameter (p < 0.001). In CF patients, IRT decreased while PAP values remained high over years. In newborns without CF, IRT decreased after birth over weeks while PAP increased within days. Conclusions The IRT × PAP product performs well as second-tier cutoff parameter for CFNBS. Screening quality parameters depend on the analytic method and on age at blood collection.