Ionization mechanisms of different lipid classes and other hydrophobic compounds have been evaluated in an ambient air laser-desorption flexible microtube plasma ionization (LD-FμTPi) setup, without sample manipulation. Lipids require a minimum laser fluency of 27 W/mm2 for efficient desorption and detection, providing the possibility for temperature-programmed laser desorption of different lipid classes. The flexible microtube plasma (FμTP) produces oxygen addition to double bonds, even to polyunsaturated molecules. The characteristic fragmentation pattern of phospholipids consisting of the neutral loss of the phosphocholine head group was verified. The formation of dimers due to hydrogen bonding and dispersion forces was observed as well. In this sense, soft ionization capabilities of the FμTP were proven in both ion modes. Ambient air mass spectrometry methods often suffer from decreased reproducibility, for instance, due to changing atmospheric conditions or sensitive positioning of the ion source. It was shown that neutrals become increasingly unstable above a distance of 7 ± 1 mm to the spectrometer's inlet, providing estimates for the free volume in LD-FμTPi MS. In this sense, no guided transport is required. The ion plume ejected from the plasma can be altered by applying a bias voltage to the copper substrate. Ions can be detected at -950 V, 300 V (negative ion mode) and -400 V, 900 V (positive ion mode), respectively. The ions are guided through an internal electric field gradient of the FμTP that arises from charged capillary walls, ideal for ion detection. In conclusion, this makes the method fast, robust, and flexible.