Tanaman sambiloto (Andrographis paniculata Nees.) merupakan salah satu tanaman obat unggulan Indonesia selain temulawak, mengkudu, pegagan, lidah buaya, lada, dan kunyit. Peningkatan produktivitas sambiloto dapat dilakukan dengan perbaikan teknik budidaya melalui sistem pemupukan. Kajian aplikasi pupuk guano terhadap pertumbuhan, produksi dan kandungan andgrographolida pada tanaman sambiloto terbatas, sehingga perlu dilakukan untuk mengetahui pengaruh pupuk guano terhadap pertumbuhan dan kandungan andrographolida pada tanaman sambiloto. Penelitian ini dilaksanakan di kebun percobaan Program Studi Agroteknologi Fakultas Pertanian Universitas Trunojoyo Madura pada bulan Januari - April 2016. Penelitian ini menggunakan Rancangan Acak Lengkap (RAL) non faktorial dengan perlakuan 6 taraf dosis pupuk guano dengan 4 ulangan. Perlakuan dosis pupuk guano per hektar meliputi G0 (control/tanpa pupuk guano), G1 (5 ton), G2 (7.5 ton), G3 (10 ton), G4 (12.5 ton), dan G5 (15 ton). Pada setiap percobaan terdapat 6 tanaman dengan rincian 3 tanaman sebagai sampel. Hasil penelitian menunjukan bahwa pemupukan guano pada dosis 15 ton/ha (G5) memberikan hasil terbaik untuk parameter tinggi tanaman, jumlah daun, luas daun, berat basah total dan berat kering total tanaman. Kandungan andrographolida (%) tertinggi diperoleh pada dosis pupuk guano 7.5 ton/ha (G2), sedangkan produksi andrograpolida (mg) tertinggi ditunjukkan oleh perlakuan G4 (dosis pupuk guano 12.5 ton/ha).Response of Growth and Production of Srographic Andrographolida Plants (Andrographis paniculata Nees) Due to Differences in Guano Fertilizer Doses Bitter plant (Andrographis paniculata Nees.) including one featured Indonesian medicinal plants in addition to ginger, noni, gotu kola, aloe vera, pepper, and turmeric. Increased productivity is bitter to do with the improvement of farming techniques through a system of fertilization. Study of guano fertilizer application on the growth, production, and content andrographolide the bitter plant is limited, so it is necessary to know the effect of guano fertilizer on the growth and content of the plant andrographolide bitter. This research was conducted at the experiment station Agrotechnology, Faculty of Agriculture, University Trunojoyo Madura at January-April 2016 by using a completely randomized design (RAL) non-factorial with 4 replications consisting of G0 (control/without guano fertilizer), G1 ( fertilizer guano 5 tons/ha) G2 (7.5 tons/ha), G3 (10 tons/ha), G4 (12.5 tons/ha) and G5 (15 tons/ha) on each trial there are 6 plants with details of 3 plants in the sample. The results showed that the guano fertilizer at a dose of 15 tons/ha (G5) can be increased plant height, leaf number, leaf area, total wet weight, and total dry weight. The highest content of andrographolide obtained at doses of 7.5 tons/ha (G2) of guano fertilizer, while the highest yield of andrographolide obtained at doses 12.5 tons/ha of guano fertilizer (G4).