To compare the cost effectiveness of adding a pancreatitis-associated protein (PAP) assay to common immunoreactive trypsinogen (IRT) and DNA cystic fibrosis (CF) newborn screening strategies. Using data collected on 553,167 newborns, PAP cut-offs were calculated based on non-inferiority of the detection rates of classical forms of CF. Cost effectiveness was considered from the third-party payer's perspective using only direct medical costs, and the unit costs of PAP assays were assessed based on a micro-costing study. Robustness of the cost-effectiveness estimates was assessed, taking the secondary outcomes of screening (ie. detecting mild forms and CF carriers) into account. IRT/DNA, IRT/PAP, and IRT/PAP/DNA strategies had similar detection rates for classical forms of CF, but the strategies involving PAP assays detected smaller numbers of mild forms of CF. The IRT/PAP strategy was cost-effective in comparison with either IRT/DNA or IRT/PAP/DNA. IRT/PAP/DNA screening was cost-effective in comparison with IRT/DNA if relatively low value was assumed to be attached to the identification of CF carriers. IRT/PAP strategies could be strictly cost-effective, but dropping DNA would mean the test could not detect CF carriers. IRT/PAP/DNA strategies could be a viable option as they are significantly less costly than IRT/DNA, but still allow CF carrier detection.