Abstract: Salbutamol (SAL) is one of the most commonly used illegal feed additive to promote growth in animal industry. Several methods have been reported for SAL detection, including immuno analytical and instrumental analysis methods. Immuno analytical analysis has high specificity and throughput in SAL detection. However, it also has limitations, such as high false positive rates, expensive antibody-costs, etc. Besides, tandem MS methods have high sensitivity and excellent repeatability, but they are usually time-consuming. Thus, rapid analysis of SAL in complex samples are still challenging. In 2015, our group developed membrane electrospray ionization (MESI). It could achieve rapid analysis of the target analytes in complex matrices with high sensitivity without sample pretreatment. In order to improve the performance of MESI, molecular imprinting technology (MIT) was explored and integrated with MESI. This new ambient ionization technique was named molecularly imprinted membrane electrospray ionization (MIM-ESI). In this study, a rapid quantitation method of molecularly imprinted membrane electrospray ionization mass spectrometry (MIM-ESI MS) was developed to detect SAL in simulated real samples directly. Experimental conditions were investigated and optimized, including membrane property, applied voltage, elution solvent, adsorption time and the template molecules. The results showed that the best adsorption time is 20 min, and the best elution solvent is methanol with 0.1% formic acid. The analyte achieved the highest intensity when applying a HV of 2.5 kV onto the MIM. The methodology had been validated, including precision, accuracy, linear range, limit of detection (LOD), limit of quantification (LOQ) and the recovery rate, which showed that this method has good sensitivity and repeatability. Through the capability of target enrichment, a 10-fold LOQ improvement for SAL standard solution could be achieved compared to the nano-electrospray ionization. Experiments for detecting simulated real samples were also carried out. SAL was spiked into urine samples directly, and the LOD and LOQ for SAL are 5 ng/L and 10 ng/L, respectively. The calibration curve of SAL in urine samples are linear over the range of 0.01-100 000 μg/L with the correlation coefficient (r²) of 0.997 6. This method doesn’t need sample pretreatment, and that is the reason why rapid analysis can be achieved (<1 min). The molecularly imprinted membrane (MIM) has function of target enrichment. It is able to achieve rapid quantitation of SAL in complex matrix at low concentration. With the capability of target enrichment, MIM-ESI MS could also be used for other applications in different fields and it is an economically and environmentally method. MIM-ESI is able to be coupled with mini-mass spectrometer (mini MS) in the future.