Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder characterized by alterations in motile cilia func-tion. The diagnosis of PCD is challenging due to the lack of standardized methods in clinical practice. High-speed video microscopy analysis (HSVA) directly evaluates ciliary beat frequency (CBF) in PCD. Recently, open-source ciliary analysis software applications have shown promise in measuring CBF accurately. How-ever, there is limited knowledge about the performance of different software applications, creating a gap in understanding their comparative effectiveness in measuring CBF in PCD. We compared two open-source software applications, CiliarMove and Cilialyzer, against the manual count method. We used videos rec-orded at high-speed of nasal ciliary brush samples from PCD patients and healthy controls. All three meth-ods showed lower median CBF values in PCD patients than healthy controls. CiliarMove and Cilialyzer identified lower CBF in PCD patients, similar to the manual count. Cilialyzer, CiliarMove and manual count demonstrated statistical significance (p-value < 0.0001) in the difference of median CBF values be-tween PCD and healthy controls. These findings support the utility of open-source software-based analysis tools. Further studies are needed to validate these findings with other genetic variants and identify the op-timal software for accurate CBF measurement in PCD.