Targeted proteomics allows the highly sensitive detection of specific peptides and proteins in complex biological samples. Here, we describe a methodology for targeted peptide quantification using a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF Pro). The prm-PASEF method exploits the multiplexing capability provided by the trapped ion mobility separation, allowing more than 200 peptides to be monitored over a 30 min liquid chromatography separation. Compared to conventional parallel reaction monitoring (PRM), precursor ions are accumulated in the trapped ion mobility spectrometry (TIMS) cells and separated according to their shape and charge before eluting into the quadrupole time-of-flight (QTOF) part of the mass spectrometer. The ion mobility trap allows measuring up to six peptides from a single 100 ms ion mobility separation with the current setup. Using these improved mass spectrometric capabilities, we detected and quantified 216 isotope-labeled synthetic peptides (AQUA peptides) spiked in HeLa human cell extract with limits of quantification of 17.2 amol for some peptides. The acquisition method is highly reproducible between injections and enables accurate quantification in biological samples, as demonstrated by quantifying KRas, NRas, and HRas as well as several Ras mutations in lung and colon cancer cell lines on fast 10 min gradient separations.