Microplastics (MPs) in the air are one of the major concerns due to their possible health risks. In addition, airborne microplastics act as a carrier for several toxic air pollutants and cause carcinogenic, mutagenic and teratogenic effects. Nylon 66 is one of the most abundant MPs in the microenvironments. In this study, we report an optimized method for the quantification of nylon 66 MPs present in the fine particulate matter (PM2.5) using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The optimized method exhibited excellent linearity, recovery rates, intraday and interday precision and sensitivity. The optimized method was deployed to carry out a pilot study to measure the mass concentrations of nylon 66 microplastics present in the inhalable fraction of particulate matter collected in a shopping complex. The observed mass concentrations of nylon 66 MPs in this study are in the range from 0.30 ng m−3 to 4.37 ng m−3. This study demonstrates the presence of inhalable fraction of nylon 66 MPs in the indoor environment and their mass concentrations were determined using HPLC-MS/MS.