The venoms of snakes are a cocktail of biochemically active substances that can have devastating effects, but also serve as template for novel drugs. Every year snakebites claim hundreds of thousands of victims, either fatally injured or maimed. Therefore, their toxins are in scientific focus and deciphering venoms is a subfield of toxinology, known as ‘venomics’. Over the centuries, venom research has evolved into an interdisciplinary branch spanning topics such as evolution, biochemistry and pharmaceutics, but also structural biology and analytics have become key elements in the modern venomics. Especially, mass spectrometry (MS) has established itself as one of the most important methods due to its continuous improvement and high versatility. This thesis deals with the composition of snake venoms and the adaptation as well as further development of modern MS techniques for toxin analysis. In the first part, the investigation of the entire Old World vipers (Viperinae) subfamily showed that a large part of their venoms and variations have hardly been described by proteomics in the literature. The toxin families found in viper venoms had a qualitative consensus and can be classified according to their relative abundance. However, compositions at the genus and species level show remarkable quantitative differences and most venoms are either protease or lipase dominated. The lack of metadata and a high variance in used quantification methods were one of the major problems for direct comparisons. Nevertheless, this chapter presents the first holistic database of all studied Old World viper venom proteomes until the beginning of 2021. In the second part of the thesis, four elapid and viper venoms were characterized for the first time in detail by proteotranscriptomics and served in the three corresponding chapters as models to investigate problems and open questions in the field of venomics, respectively. The venom of the Anatolian meadow viper (Vipera anatolica senliki), like most vipers, contains toxins of high molecular weight that cannot be detected intact until now. Current top-down approaches to measure proteins directly from the crude venom are limited to about 30 kDa and thus only partially useful for viper venoms. With an alternative in-source decay (ISD) using 1,5-diaminonaphthalene (DAN), this limit could be increased to 70 kDa. The applied matrix-assisted laser desorption/ionization (MALDI) made it also possible to de novo annotate and identify various toxin families and proteoforms even in parallel in a sample. The following chapter highlights that current venomics approaches only allow relative quantifications, as different toxins would require dozens of different standards for absolute values according to classical methods, which is practically impossible. However, many snake toxins contain sulfur, mostly in structurally relevant disulfide bridges, which can be quantified in comparison to a single sulfur standard of known concentration. The venom atomization by an inductively coupled plasma (ICP) allows the elemental sulfur content to be accurately measured by MS. With a known sequence, the concentrations of the individual toxins could be exactly determined. In combination with three parallel bottom-up and top-down protocols, this hybrid approach of molecular and elemental MS enabled the absolute quantification and detailed characterization of venom proteomes using of desert cobras (Walterinnesia) as example. The last chapter deals with a more biological aspect of the venom apparatus, as little is known about the tissue-internal processes of venom production and storage. For this purpose, fixed venom gland tissue of the Egyptian cobra (Naja haje) was scanned using MALDI. The high two-dimensional resolution in tenth micrometer range allows the detection of spatially resolved spectra of toxin-specific m/z masses. This MS imaging (MSI) of in situ toxin mapping by spatial intensity maps across a venom gland showed that the different families and individual toxins are produced only in specific areas and are not ubiquitous in the gland.