Leishmaniasis a group of parasitic diseases related to a major health problem in Asian sub-continent, Africa and rest of world. New discovery and isolation of drug against different types of leishmaniasis is one of the researcher priorities. This works describes an analytical platform based on a semi-high resolution Antileishmanial bioassays in combination with hyphenation of high performance liquid chromatography, solid phase extraction, and tube-transfer nuclear magnetic resonance spectroscopy. i.e., HPLC-SPE-ttNMR/semi-high resolution Antileishmanial assay. The platform enables fast screening for individual leishmania tropica inhibitory analytes in complex matrices, followed by structural identification targeted these L. tropica inhibitors via in vitro, in vivo and cytotoxic test. The plant Lawsonia Inermis was collected from territory of Dera Ismail Khan. Methanol extracts of plant material at concentrations of 10 - 100 µg/ml were tested for them in vitro anti-leishmanial effects on L. tropica KWH23 promastigotes for 24-48 h, relative to negative control and Amphotericin-B (standard drug). For in vivo anti-leishmanial activity, the extract was tested against L. tropica-infected albino mice, while cytotoxicity was investigated against mammalian cells (lymphocytes). For Lawsonia Inermis leaves, mean % inhibition of extracellular promastigotes at 10 µg/ml, 25 µg/ml, 50 µg/ml, and 100 µg/ml after 48 h were 98.2 ± 0.06, 98.75 ± 1.09, 99.31 ± 0.00 and 100.00 ±0.00 respectively. After 8 weeks, mean lesion size decreased from 0.8 ± 0.2mm to 0.3 ± 0.1mm (p < 0.01), and % cure at 150mg/kg against intracellular amastigotes in albino mice was 97.02 (95% C.I = 96.14-98.10). IC50 for Lawsonia Inermis leaves was 12.22 µg/ml (95% C.I = 11.54-13.84) against lymphocytes. The results obtained in this study show that leaves of Lawsonia Inermis are safe and possess potent anti-leishmanial activity. Fractional separation of Lawsonia Inermis leaves was done by applying polarity based solvent system comprised of n-Hexane, Chloroform, Ethyl acetate and Methanol. Phytochemical screening declared that Flavonoids, Coumarins, Phenol and Alkaloids are present in Lawsonia Inermis. Further characterization including identification and evaluation of isolated fractions and compounds was performed via hyphenated HPLC-HRMS-SPE-ttNMR spectroscopic techniques. Active analytes were cumulatively trapped on SPE cartridges and the structures identified by 1H NMR experiments obtained in the HPLC-SPE-ttNMR mode showed six known compounds from the leaves of Lawsonia Inermis; Luteolin (1), Lalioside (2), Luteolin-4’-O-β-D-glucopyranoside (3), Apigenin 4-O-beta-D-glucopyranoside(4), Apigenin (5) and 2, 4, 6-trihydroxyacetophenone-2-O-β-D-glucopyranoside (6). In vitro results showed that luteolin having IC50 of 4.15 value; eliminate the clinically isolated L. tropica promastigotes within 48 hours which is a promising result against leishmaniasis while Amphotericin-B was the reference drug. Lesions sizes were determined in BALB/c mice when luteolin were given to mice while proceeding in vivo study. After 60 days’ treatment, most of mice in Group I, II, III, IV and V were recovered; lesions of few mice were completely healed at 90th day in same groups. In negative control study, 1 mouse died on 45th day and 2 on 90th day. For compound 1, mean lesion size decreased from 0.82 ± 0.12 up to 0.10 ± 0.01 after 120 days, with 97% cure of intracellular L. tropica amastigotes at dose 15mg/kg, compared to Amphotericin B which had 95% cure at a dose of 30 mg/kg. IC50 for compound 1 was 4.15 µg/ml against lymphocytes. The results indicate that luteolin is a potent inhibitor of L. tropica promastigotes and amastigotes, but it had higher cytotoxic effects against lymphocytes, relative to luteolin-4’-O-β-D-glucopyranoside. To eliminate leishmaniasis through discovery of potent drug, further research work should be done to check the efficacy of luteolin and luteolin-4’-O-β-D-glucopyranoside on dogs. Next step should be monkeys and human beings later on.