Classically dissecting the role and locale of critical enzymatic steps in SL biosynthesis and assessing the effect on the parasite fitness and virulence could turn into an overwhelmingly challenging task aggravated by: the complexity of the metabolic pathway itself; the ability of the parasite to salvage (Coppens, 2013), hijack and remodel host SL; and developmental regulation during the parasitic life cycle, which adds another layer of intricacy rendering the deconvolution of any observed effects difficult to interpret. Fortunately, many of those problems can be now overcome with advances in technology. High resolution localization studies in protozoan parasites can benefit greatly from new microscopic techniques such as Airy-scan (Huff, 2015), super-resolution microscopy (Florentino et al. 2014) and upcoming technologies, e.g. phase-modulation nanoscopy (Pal, 2015; Ward & Pal, 2017), which can elucidate spatial arrangement of proteins of interest within the parasite to reveal potential interaction partners and shed light on mechanistic features. Similarly, new advances in chemical probes, and SL analogues in particular, such as bifunctional lipid technology (Haberkant & Holthuis, 2014) coupled with high throughput proteomic (Ramaprasad et al. 2015), could identify different interaction partners that would help map the biosynthetic pathway and its critical interactions. The effects of these probes on the parasite (and host) cell can now be comprehensively evaluated by monitoring the transcriptome, proteome, metabolomics (Watson, 2010) and lipidome (Marechal et al. 2011). Such studies could reveal multiple windows of opportunity to exploit as potential drug targets. The targets identified in this way can now be rapidly genetically validated in the parasitic protozoa by applying modern gene editing technologies, such as CRISPR/Cas9 (Sugi et al. 2016). Compared with the classical methodologies, this tool enables fast and efficient application for single gene (Serpeloni et al. 2016), and systematic genome-wide knockout generation (Sidik et al. 2016). Additionally, the development of novel orthogonal approach for conditional knockout strategies, e.g. tetracycline-induced gene disruption Tet-system (Meissner et al. 2002), rapamycin-induced Cre recombinase-assisted gene excision (Andenmatten et al. 2013; Collins et al. 2013; Jimenez-Ruiz et al. 2014), has allowed testing of essential gene functionality, in Leishmania spp. (Duncan et al. 2016) and T. gondii (Pieperhoff et al. 2015).