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Mica exhibits a relatively high Rb/Sr ratio and possesses a closure temperature for the Rb-Sr isotope system that is higher than the ore-forming temperature range of low- to medium-temperature deposits. Consequently, utilizing the laser ablation (LA) technique for in situ Rb-Sr dating in mica constitutes a significant methodology for resolving the mineralization chronology of low- to medium-temperature hydrothermal ore deposits. In this study, we employed a combination of a triple quadrupole inductively coupled plasma mass spectrometer (ICP-MS/MS) and 193 nm LA system with SF6 as the reaction gas to achieve interference removal of 87Rb+ on 87Sr+. We then developed an in situ Rb-Sr dating method for mica. In addition, an investigation was conducted to identify the factors that could influence the analytical accuracy of the method. Subsequently, we applied the developed method to carry out in situ Rb-Sr dating for the ZBH-25 biotite, a national primary reference material (GBW04439) for K-Ar dating, and yielded a younger age (ca. 12%, relative standard deviation, RSD) than the reference K-Ar age when using NIST SRM 610 as the reference material for external calibration. This finding indicated that in the process of determining Rb-Sr ages for mica by LA-ICP-MS/MS, there are significant matrix effects between the NIST SRM 610 and the mica samples, resulting in inaccurate Rb-Sr ages. In response to this issue, a two-step calibration method is proposed here. Based on external calibration using NIST SRM 610, ZBH-25 biotite is employed as a second external reference material to perform a second calibration of the sample data to correct the matrix effect between the glass standards and the natural mica samples, thereby improving the accuracy of Rb-Sr dating by LA-ICP-MS/MS. Using the proposed method, in situ Rb-Sr dating by LA-ICP-MS/MS was performed on mica samples from monzonite exposed in Mt. Dromedary, New South Wales, Australia, porphyritic granodiorite in Fangshan pluton and albionite granite in the Gyirong area of Tibet. The obtained Rb-Sr age results show agreement with the recommended values within the analytical uncertainty, which serves to validate the accuracy and effectiveness of the proposed method. [ABSTRACT FROM AUTHOR]