High-throughput and efficient identification of drugs in a complex mixture is required in many pharmaceutical industries and drug testing laboratories. The present study aims to utilize an efficient pooling strategy for the preparation of high-resolution tandem mass spectrometry (HR-MS/MS) library of drugs with different therapeutic properties. Compounds were classified into four classes of low, medium–low, medium–high and high log P values and were pooled into five pools (100 drugs pool). Compounds with isobaric and close log p values were placed in separate groups to overcome the co-elution problem. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) was utilized to generate a high-resolution tandem mass spectrometry (MS/MS) spectral library in both positive and negative electrospray ionization modes of 491 drugs. Spectra were collected by LC-MS/MS analysis using system automated collision energy i.e. of 25–60 eV and four predetermined collision energies (10, 20, 30 and 40 eV) for each compound using schedule precursor list of [M + H]+ and [M + Na]+ ions. To validate the applicability of the library, serum samples spiked at three concentrations close to bioavailability (0.308 ng/mL, 3.08 ng/mL and 30.08 ng/mL) of drugs and were analyzed by LC-QTOF-MS/MS. The HRMS library search successfully identified all compounds present in the spiked serum samples based on exact masses of precursor ions, MS/MS data, and retention time etc. The accurate mass LC-QTOF-MS based tandem mass spectral libraries represent a useful tool for the identification of drugs in clinical samples, for pharmacological and forensic screening applications etc.