In research based sciences “Alternatives to Animals” can be stated as testing methods which can replace partial or absolute use of animals; and this field not merely rely on the replacement of tests but the development and implementation of those testing methods to avoid the use of live animals also comes under this section. There are two major alternatives to in-vivo animal testing: The first ones are in vitro cell culture techniques, the seconds ones are in silico computer simulations. Microdosing is one of the other alternative options to study the basic behaviour of drugs by using lower than expected doses to produce whole body effects in volunteer human beings. Microfluidic chips are getting key interest in alternatives; because of the provision of more complex information as compare to other in vitro tests. Imaging studies like computed tomography (CT), magnetic resonance imaging (MRI), functional magnetic resonance imaging (fMRI), positron emission tomography (PET) and single photon emission computed tomography (SPECT) are playing unmatchable role while studying some organ systems of the body. Non-Animal Method Database resources are gaining much more popularity since the concept of alternative to animals is culminating all around the world. Because of the advancement in molecular and cellular biology experiments; a lot of information is being generated and stored for in vitro and in silico experiments for better scientific understandings of experimental drugs on body systems. Three Rs (3Rs) Replacement, Reduction and Refinement of Russell and Burch are like guiding principles for more ethical use of animals in testing. In this review, main alternative methods to animal studies are summarized.