AbstrakPiper betle L. var Nigra (sirih hitam) merupakan salah satu jenis tanaman obat yang berpotensi untuk dikembangkan dengan metode kultur jaringan. Penelitian ini bertujuan untuk mengetahui pengaruh variasi zat pengatur tumbuh Indole Acetic Acid (IAA), Benzyl Amino Purine (BAP), Kinetin terhadap senyawa metabolit sekunder yang terdapat kalus sirih hitam. Eksplan daun dari sirih hitam ditanam pada medium Murashige dan Skoog dengan 4 perlakuan kombinasi (I1,0K1,0; I1,0K1,5; I1B1,5; I0,5B0,5). Kalus dipelihara selama 8 minggu. Simplisia kalus sirih hitam di maserasi menggunakan pelarut metanol dan diidentifikasi menggunakan Gas Chromatography Mass Spectra (GCMS). Berdasarkan analisis GCMS menunjukkan bahwa senyawa metabolit sekunder dengan presentase tertinggi terdapat pada masing- masing perlakuan yaitu senyawa gamma sitosterol (14,88%) pada perlakuan I1,0K1,0 ; senyawa 14-Beta H Pregna (15,94%) pada perlakuan I1,0K1,5 serta senyawa beta-d-glucopyranoside (15,54%) pada perlakuan I0,5B0,5; dan senyawa beta-d-glucopyranoside (5,63%) pada perlakuan I1,0 B1,5. Katakunci: Kalus, Piper betle L. var Nigra, senyawa metabolit sekunder AbstractPiper betle L. Var Nigra (black betel) is one type of medicinal plant that has the potential to be developed by tissue culture method. This study was aims to determine the effect of variations in growth regulator Indole Acetic Acid (IAA), Benzyl Amino Purine (BAP), and Kinetin on secondary metabolite compounds contained in black betel callus. The explant of black betel’s leaf was cultured on Murashige and Skoog medium with four combination treatments (I1.0K1.0; I1.0K1.5; I1B1.5; I0.5B0.5). The callus was maintained for eight weeks. Simplisia of black betel’s callus was macerated using methanol solvent and identified using Gas Chromatography Mass Spectra (GCMS). GCMS analysis showed that secondary metabolites with the highest percentage in each treatment were gamma sitosterol compound (14.88%) in treatment I1.0K1.0; 14-Beta H Pregna compound (15.94%) in I1.0K1.5 treatment and beta-d-glucopyranoside compound (15.54%) in I0.5B0.5 treatment; and beta-d-glucopyranoside compound (5.63%) in I1.0 B1.5 treatment.Keywords: Callus, Piper betle L. Var Nigra, secondary metabolite compounds