Nonacog beta pegol (N9-GP; Refixia ®; Novo Nordisk Pharma Ltd.) is a glycoPEGylated extended half-life (EHL) recombinant factor IX (rFIX) developed for prophylaxis and treatment of bleeding episodes in patients with hemophilia B. The efficacy and tolerability of nonacog beta pegol were confirmed in phase 3 clinical trials. Post-administration monitoring of nonacog beta pegol activity may be necessary in specific clinical scenarios. In this two-part study, we evaluated nonacog beta pegol recovery using one-stage clotting assay activated partial thromboplastin time (aPTT) reagents available in Japan, and tested this laboratory analysis at three field sites using various analyzers. Pooled congenital hemophilia B plasma was spiked with nonacog beta pegol. All samples were verified for accurate spiking. Nonacog beta pegol was accurately measured using the ACL TOP®550 coagulation analyzer (Instrumentation Laboratory, Holliston, USA) and ThromboCheck APTT-SLA, ThromboCheck APTT(S), ThromboCheck APTT, Coagpia®APTT-N and Coagsearch®APTT, aPTT reagents readily available in Japan. The CS-5100 analyzer (Sysmex Corporation, Kobe, Japan) using ThromboCheck APTT-SLA underestimated nonacog beta pegol. CS and CA series analyzers (Sysmex Corporation) using Data-fi APTT accurately recovered nonacog beta pegol. Three of the aPTT reagents tested (PTT LA FR, STA®-PTT A and Platelin L II) overestimated FIX activity and are unfit for post-administration monitoring.