For decades antimonials were the drugs of choice for the treatment of visceral leishmaniasis (VL), but the recent emergence of resistance has made them redundant as first-line therapy in the endemic VL region in the Indian subcontinent. The application of other drugs has been limited due to adverse effects, perceived high cost, need for parenteral administration and increasing rate of treatment failures. Liposomal amphotericin B (AmB) and miltefosine (MIL) have been positioned as the effective first-line treatments; however, the number of monotherapy MIL-failures has increased after a decade of use. Since no validated molecular resistance markers are yet available, monitoring and surveillance of changes in drug sensitivity and resistance still depends on standard phenotypic in vitro promastigote or amastigote susceptibility assays. Clinical isolates displaying defined MIL- or AmB-resistance are still fairly scarce and fundamental and applied research on resistance mechanisms and dynamics remains largely dependent on laboratory-generated drug resistant strains. This review addresses the various challenges associated with drug susceptibility and -resistance monitoring in VL, with particular emphasis on the choice of strains, susceptibility model selection and standardization of procedures with specific read-out parameters and well-defined threshold criteria. The latter are essential to support surveillance systems and safeguard the limited number of currently available antileishmanial drugs.