Holographic Optical Tweezers (HOT) is a technique in which the phase of trapping laser is modulated for generating steerable, multiple optical traps in a sample chamber. An indigenously developed HOT set-up at Raja Ramanna Centre for Advanced Technology, Indore has been used to trap thermo-responsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAM-co-AAc) spherical particles of 1.6 μm diameter suspended in aqueous medium. The videos of the trapped particles were digitally processed to track the particle positions as a function of time. From these measurements lateral trap stiffness for pNIPAM-co-AAc particles was determined as a function of trap power and temperature using Equipartition and Boltzmann Statistics methods. Both the methods gave similar results and the value for the trap stiffness at 25°C with trapping laser power of 33 mW was estimated to be 0.14±0.01μN/m. Since the optical trap stiffness depends on particle size and refractive index which vary as a function of temperature the variation of the measured optical trap stiffness as a function of temperature could be used to determine the volume phase transition of the thermo?responsive microgel particles. The results should also be useful in investigating the interaction between pNIPAM-co-AAc particles trapped in different lattice configurations that can be generated using HOT