Mass spectrometry (MS) is an extremely powerful analytical technique, which plays a central role in many scientific fields. The Heaney research group critically reviewed the 2021 applications of ambient ionization mass spectrometry (AIMS), as a follow-up to the previous year's review paper. 1 AIMS enables the surface analysis of samples in their native environment in a high-throughput fashion and is widely used in disease diagnostics, forensics, homeland security applications, and the environmental sciences. Heaney et al. provide an overview of ambient ionization techniques and highlight the applicability in a wide range of scientific fields, either carried out in the laboratory or out in the field. High-performance liquid chromatography (HPLC) hyphenated to MS is the method of choice to profile oligonucleotide therapeutics. Hannauer et al. summarized the advancements in separation science considering ion-pairing reversed-phase chromatography, hydrophilic interaction chromatography, and two-dimensional liquid chromatography. In particular, the effect of chromatographic elution conditions and column chemistries on retention, resolving power, and MS compatibility are outlined. In addition, recent software developments for the tandem MS analysis of oligonucleotides are discussed. Minkus, Bieber, and Letzel provided a detailed review of the processing of mass-spectrometric non-target screening (NTS) data. NTS is an untargeted comprehensive analysis methodology based on high-resolution MS, where the entire mass range of small organic molecules of anthropogenic origin is recorded within a very short cycling time, leading to complex and large data sets. In particular, Minkus et al. review major contributions that concern the processing of NTS data, prioritization of features, as