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Introduction: Robot-assisted navigation bronchoscopy (RANB) has been gaining traction as a new technology for minimally invasive biopsies of peripheral pulmonary lesions (PPLs). Cryobiopsy is an established method of procuring satisfactory lung tissues and can be safely paired with RANB. While some studies have evaluated the diagnostic accuracy and yield of this procedure, there is limited data on the utility of various biopsy tools, the sequence of use and differences in tissue characteristics based on the sampling techniques. Therefore, this study aims to examine the real-life performance of needle, forceps and cryoprobe when utilized in succession with RANB in the biopsy of PPLs, and to evaluate the specific types of tissue samples obtained from each instrument., Methods: In a single-center retrospective study, 50 patients presenting 52 PPLs underwent biopsies sequentially using fine-needle aspiration (FNA), forceps, and cryoprobe. All procedures were performed via cone-beam CT-assisted RANB. Performance metrics, such as diagnostic yield, sensitivity, and specificity, were determined by classifying malignancy as true positives and explicit benign findings as true negatives. Tissue sizes and subtypes were based on pathology description and compared with Student's t test and χ2, respectively., Results: Cryobiopsy, when performed sequentially after FNA then forceps, was able to retrieve more alveolar tissue (p = 0.0098) among diagnosed lesions and showed higher diagnostic yield (p = 0.001) in PPL biopsy with RANB platform. Cryobiopsy was also able to obtain larger sample size (p = 0.0087). No difference was observed in the diagnosis of malignancy between forceps and cryobiopsy (p = 0.8877)., Conclusion: The integration of RANB and sequential biopsies, utilizing FNA, forceps, and cryoprobe, can efficiently diagnose PPLs and procure alveolar tissues. Further research based on histopathological subtypes is required to assess its prognostic significance., (© 2023 S. Karger AG, Basel.)