Loss analysis due to the insertion of optical fiber in short wavelength spectrophotometer system is experimentally conducted to improve resolution and flexibility of the system design. In this paper, type, length and bending diameter of the optical fiber are analyzed as a preliminary work towards the development of a low-loss spectrophotometer system. In order to analyze a suitable type of fiber to be adopted in this system, a 20 mm length and 0 mm bending diameter of a single-mode fiber (SMF), multi-mode fiber (MMF) and Polymethyl methacrylate (PMMA) fiber are tested. The length and bending diameter of the suitable fiber is then varied from 20 mm to 60 mm and 6 mm to 22 mm respectively. In this paper, light emitting diode (LED) centered at 525 nm wavelength with 35 nm full width half maximum (FWHM) is used as the spectrophotometer light source while silicon photodiode is used as the detector. In this work, photodetector output voltage is recorded to analyze the loss contributed by these three parameters. At this particular wavelength, PMMA is found to be a suitable fiber to be adopted due to its minimal loss performance. Besides the fiber type, having a minimal fiber length with maximal fiber bending diameter can reduce loss due to the insertion of optical fiber in spectrophotometer system, hence improving the spectrophotometer resolution performance.