Bioorthogonal reactions, due to its excellent selectivity and time-efficiency, have emerged as a popular tool for protein and cell probing. Among all the bioorthogonal reactions, the inverse electron-demand Diels–Alder reaction (IEDDA) reaction has its advantage of bearing the fastest kinetics. Although the IEDDA reaction drew considerable attention in chemical biology in the last decade, challenges lie in finding the suitable dienophiles. Strained dienophiles, for example, trans-cyclooctene derivatives, can undergo ultrafast IEDDA reactions and therefore have been extensively developed. Unstrained alkenes and alkynes, however, have not been well investigated as IEDDA handles. In general, unstrained dienophiles are more straightforward to synthesise compared with strained dienophiles, therefore they are more accessible to researchers. In addition, the absence of a highly reactive bond makes unstrained dienophiles inert to biological nucleophiles, which allows effectively cellular labelling. In this dissertation, I described three different unstrained dienophiles for different biological purposes. Allyl handle is thiol-stable and non-toxic, which was utilised to label apoptotic cells in a pre-targeting manner. Enol ethers can react with tetrazines to decage protected amino acids and prodrugs. Potassium arylethynyltrifluoroborate, as a novel dienophile, was shown to react fast with pyridyl tetrazines controllably and this new IEDDA was applied to label proteins site-selectively and to fluorescently label two proteins orthogonally. In addition to IEDDA reactions, other bioorthogonal reactions were also developed using these versatile unstrained handles. Allyl-bearing amino acids and proteins can undergo an acetophenone-mediated hetero-[2+2] photocycloaddition with maleimide derivatives, expanding the toolbox of photo-triggered chemistry for protein modification. The potassium arylethynyltrifluoroborate handle was also found reactive in copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) and showcased the huge potential for protein labelling and multicolour cellular labelling.